Rapid epidemiologic typing of bacteria

ABSTRACT

Methods for typing a strain of an organism are provided, the methods comprising the steps of amplifying, in a single reaction mixture containing nucleic acid from the organism, dividing the reaction mixture into a plurality of sets of second-stage reaction wells, each set of second-stage reaction wells containing a different pair of second-stage primers, subjecting each of the second-stage reaction wells to amplification conditions to generate a plurality of second-stage amplicons, melting the second-stage amplicons to generate a melting curve for each second-stage amplicon, and identifying the strain of the organism from the melting curves.

PRIORITY

This application is a continuation application of, and claims priorityto, U.S. application Ser. No. 12/467,654 filed on May 18, 2009 andissued as U.S. Pat. No. 9,200,329 on Dec. 1, 2015, which claims priorityto U.S. Provisional Patent Application Ser. No. 61/054,357 filed on May19, 2008, and titled Rapid Epidemiologic Typing of Bacteria, the entirecontents of each of which are incorporated by reference herein.

GOVERNMENT INTEREST

This invention was made with government support under Grant Nos. U01AI061611, K12 HD001410, and R43 AI063695 awarded by National Institutesof Health. The government has certain rights in the invention.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 1267-6TSCT_ST25.txt, 17,396 bytes in size, generated onJan. 20, 3016 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is incorporated by reference into thespecification for its disclosures.

BACKGROUND OF THE INVENTION

In the United States, Canada, and Western Europe infectious diseaseaccounts for approximately 7% of human mortality, while in developingregions infectious disease accounts for over 40% of human mortality.Infectious diseases lead to a variety of clinical manifestations. Amongcommon overt manifestations are fever, pneumonia, meningitis, diarrhea,and diarrhea containing blood. While the physical manifestations suggestsome pathogens and eliminate others as the etiological agent, a varietyof potential causative agents remain, and clear diagnosis often requiresa variety of assays be performed. Traditional microbiology techniquesfor diagnosing pathogens can take days or weeks, often delaying a propercourse of treatment.

In recent years, the polymerase chain reaction (PCR) has become a methodof choice for rapid diagnosis of infectious agents. PCR can be a rapid,sensitive, and specific tool to diagnose infectious disease. A challengeto using PCR as a primary means of diagnosis is the variety of possiblecausative organisms and the low levels of organism present in somepathological specimens. It is often impractical to run large panels ofPCR assays, one for each possible causative organism, most of which areexpected to be negative. The problem is exacerbated when pathogennucleic acid is at low concentration and requires a large volume ofsample to gather adequate reaction templates. In some cases there isinadequate sample to assay for all possible etiological agents. Asolution is to run “multiplex PCR” wherein the sample is concurrentlyassayed for multiple targets in a single reaction. While multiplex PCRhas proved to be valuable in some systems, shortcomings exist concerningrobustness of high level multiplex reactions and difficulties for clearanalysis of multiple products. To solve these problems, the assay may besubsequently divided into multiple secondary PCRs. Nesting secondaryreactions within the primary product increases robustness. However, thisfurther handling can be expensive and may lead to contamination or otherproblems.

Similarly, immuno-PCR (“iPCR”) has the potential for sensitive detectionof a wide variety of antigens. However, because traditional ELISAtechniques have been applied to iPCR, iPCR often suffers fromcontamination issues that are problematic using a PCR-based detectionmethod.

The present invention addresses various issues of contamination inbiological analysis.

SUMMARY OF THE INVENTION

Accordingly, methods for identifying an organism or typing a strain ofthe organism are provided, comprising the steps of: (a) obtaining asample of the organism, (b) amplifying, in a single reaction mixturecontaining nucleic acid from the organism, a plurality of first-stageamplicons using pairs of first-stage primers, the pairs of first-stageprimers designed to hybridize to genomic regions of the organism thatare specific to that organism, (c) dividing the reaction mixture into aplurality of sets of second-stage reaction wells, each set ofsecond-stage reaction wells containing a different pair of second-stageprimers, (d) subjecting each of the second-stage reaction wells toamplification conditions to generate a plurality of second-stageamplicons, (e) melting the second-stage amplicons to generate a meltingcurve for each second-stage amplicon, and (f) identifying the organismor strain from the melting curves.

In another illustrative aspect, methods for identifying a plasmid-bornegenes in an organism are provided, comprising the steps of (a) obtaininga sample of the organism, (b) amplifying, in a single reaction mixturecontaining nucleic acid from the organism, a plurality of first-stageamplicons using pairs of first-stage primers, the pairs of first-stageprimers designed to hybridize to plasmid-based sequences, (c) dividingthe reaction mixture into a plurality of sets of second-stage reactionwells, each set of second-stage reaction wells containing a differentpair of second-stage primers, (d) subjecting each of the second-stagereaction wells to amplification conditions to generate a plurality ofsecond-stage amplicons, (e) melting the second-stage amplicons togenerate a melting curve for each second-stage amplicon, and (f)identifying the strain of the organism from the melting curves.

Additional features of the present invention will become apparent tothose skilled in the art upon consideration of the following detaileddescription of preferred embodiments exemplifying the best mode ofcarrying out the invention as presently perceived.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flexible pouch according to one embodiment of thisinvention.

FIG. 2A shows an embodiment of the cell lysis zone of the flexible pouchaccording to FIG. 1.

FIG. 2B shows an embodiment of a portion of a bladder corresponding tothe cell lysis zone shown in FIG. 2A.

FIG. 2C shows an embodiment of the cell lysis zone of the flexible pouchaccording to FIG. 1 having an alternative vortexing mechanism.

FIG. 3 shows an embodiment of the nucleic acid preparation zone of theflexible pouch according to FIG. 1.

FIG. 4 shows an embodiment of the first-stage amplification zone of theflexible pouch according to FIG. 1.

FIG. 5A is similar to FIG. 1, except showing an alternative embodimentof a pouch.

FIG. 5B is a cross-sectional view of the fitment of the pouch of FIG.5A.

FIG. 5C is an enlargement of a portion of the pouch of FIG. 5A.

FIG. 6A is a perspective view of another alternative embodiment of apouch.

FIG. 6B is a cross-sectional view of the fitment of the pouch of FIG.6A.

FIG. 7 shows illustrative bladder components for use with the pouch ofFIG. 6A.

FIG. 8 is an exploded perspective view of an instrument for use with thepouch of FIG. 6A, including the pouch of FIG. 6A.

FIG. 9 shows a partial cross-sectional view of the instrument of FIG. 8,including the bladder components of FIG. 7, with the pouch of FIG. 6Ashown in shadow.

FIG. 10 shows a partial cross-sectional view of the instrument of FIG.8, including various bladders for pinch valves and the pouch of FIG. 6A.

FIG. 11 shows amplification curves from second-stage amplification of asample that was lysed and amplified in a pouch of FIG. 5A (

positive control;

S. cerevisaie target 1;

S. cerevisaie target 2;

S. cerevisaie target 3;

S. pombe target 1;

S. pombe target 2;

negative controls).

FIG. 12A is similar to FIG. 6A, except showing a pouch having asecond-stage high density array.

FIG. 12B shows a modification of a component of the instrument of FIG.8. A support member has been provided with a motor configured for usewith the pouch of FIG. 12A.

FIG. 13 is an exploded perspective view of the second-stage high densityarray of FIG. 12A.

FIG. 14 is a bottom view of the second-stage high density array of FIG.12A, shown during construction of the second-stage high density array.

FIGS. 15A-15C show identification of Gram-negative organisms using rpoBand gyrB. Outer first-stage amplicons were generated from 5 organisms(P. aeruginosa (Pa, (

)), E. coli (Ec, (

)), K. pneumoniae (Kp,

)), K. oxytoca (Ko,

)) and H. influenzae (Hi,

) with degenerate outer primers targeting the rpoB and gyrB genes. Outerfirst-stage amplicons were nested into a set of organism-specific innersecond-stage primers with the resulting real time amplification curvesshown.

FIG. 15A shows that second-stage primers specific for either the rpoB orgyrB gene of Pa amplify only from the Pa template, except for a minoramplification with Ec in late cycles.

FIG. 15B shows that the rpoB “enteric” second-stage primer amplifies Ec,Kp and Ko, but for the gyrB gene a specific primer targets only Kp.

FIG. 15C shows that second-stage primers specific for either the rpoB orgyrB gene of Hi amplify only from the Hi template, except for a minoramplification with Ko near the end of the reaction.

FIGS. 16A-16D show Identification of Gram-positive organisms using rpoB,with baseline subtracted amplification curves. Outer first-stageamplicons were generated from 4 organisms (S. pneumoniae), S.agalactiae, S. aureus, and L. monocytogenes) with degenerate outerfirst-stage primers targeting the rpoB gene. Outer first-stage ampliconswere nested into a set of organism-specific inner primers with theresulting real time amplification curves shown. Second-stage primersspecific for the rpoB gene of Sp (FIG. 16A), Sag (FIG. 16B), Sa (FIG.16C), or Lm (FIG. 16D) have significant amplification curves only fromthe appropriate templates. Minor amplification curves with othertemplates are under investigation and likely result from the formationof “primer dimers” (especially in the case of the S. aureus primers) orpossibly cross-amplification.

FIGS. 17A and 17B show melting profiles of amplicons from theamplification reactions shown in FIGS. 15A and 15B. The melting profileof each amplicon was generated using the dye LCGreen® Plus. Ampliconsmelted with characteristic Tms that ranged from 78° C. for H. influenzaeto 90° C. for P. aeruginosa. FIG. 17A shows the results from the rpoBgene, while FIG. 17B shows the results from the gyrB gene.

FIGS. 18A and 18B show results for N. meningitidis. FIG. 18A showsamplification of the gyrB gene, while FIG. 18B shows the results for therpoB gene for this target.

FIGS. 19A-19D show differentiation of enteric organisms usingpreferential primers in rpoB amplification. FIG. 19A shows amplificationusing the “pan-enteric” primers, FIG. 1913 shoes amplification using E.coli preferential primers, FIG. 19C shows amplification using K.pneumoniae primers, and FIG. 19D shows amplification using E. cloacaeprimers.

FIGS. 20A and 20B show a decision tree for enteric identification basedon Cp fingerprint. FIG. 20A is a table showing illustrative Cp cut-offvalues, and FIG. 20B shows a flow-chart using the values of FIG. 20A.

FIGS. 21A and 21B show superimposed melting curves from 13 patients wasanalyzed for heterozygous changes in the MCAD gene using theLightScanner® (Idaho Technology). Results at two exons are shown todemonstrate how single base-pair changes are analyzed by heteroduplexmelting. FIG. 21A shows the results for exon 1, in which all patientswere homozygous for a single allele; no heteroduplexes were formedduring melting and all curves matched the control. FIG. 21B shows exon5, in which one patient had a heterozygous single nucleotide change fromT to C, easily observable here by the early melting heteroduplex (curveshifted to the left).

FIG. 22 is a simplified schematic of how the second-stage for straintyping. The top well does not have a standard so that the unknown isrepresented alone. In the second panel, the outer amplicon from unknown“X” enters the array and is distributed to all wells. In the uppermostwell, X alone is amplified; in the lower wells, X and a standard alleleare amplified together. After PCR, the amplicons undergo high-resolutionmelt analysis, with curves normalized to the melting curve of X. Where Xand the standard allele are different, heteroduplexes form, and the meltdoes not match that of X. Where X and the standard are the same, themelting curves match, and the allele is identified.

FIGS. 23A and 23B show results for four S. aureus clinical isolates (3MRSA and 1 MSSA), identified by melt to represent 3 different alleles ofaroE, which were used to demonstrate the feasibility of using highresolution melting and heteroduplex analysis for MLST. One MRSA isolatewas designated as the unknown (X). Two other MRSA isolates (A and C) andone MSSA isolate (B) represent the “standard” alleles. FIG. 23A showsmelting curves normalized by the LightScanner software to highlightdifferences. When compared in this manner, the melting curves of A+X andB+X are shown to be different from the melting curve of X alone. C+X,however, has exactly the same melting properties as X alone, suggestingthat these isolates have the same sequence at this locus (alleleX=allele C). FIG. 23B shows a “difference plot” generated by theLightScanner software and used to magnify melting curve differences foreasier visualization. In this type of analysis, one melting curve isdesignated as the reference allele, and the difference in fluorescenceof the reference and another allele are plotted at each temperature. Inthe difference plot shown here, the unknown X is designated as thereference, and relative differences are shown. Melting curves from A+Xand B+X again show significant differences when compared to X alone; nodifference is seen in C+X vs X alone, suggesting that X and C are highlysimilar, if not identical, in sequence.

DETAILED DESCRIPTION

The self-contained nucleic acid analysis pouches described herein may beused to assay a sample for the presence of various biologicalsubstances, illustratively antigens and nucleic acid sequences,illustratively in a single closed system. In one embodiment, the pouchis used to assay for multiple pathogens. Illustratively, various stepsmay be performed in the optionally disposable pouch, including nucleicacid preparation, primary large volume multiplex PCR, dilution ofprimary amplification product, and secondary PCR, culminating withreal-time detection and/or post-amplification analysis such asmelting-curve analysis. It is understood, however, that pathogendetection is one exemplary use and the pouches may be used for othernucleic acid analysis or detection of other substances, including butnot limited to peptides, toxins, and small molecules. Further, it isunderstood that while the various steps may be performed in pouches ofthe present invention, one or more of the steps may be omitted forcertain uses, and the pouch configuration may be altered accordingly.

While PCR is the amplification method used in the examples herein, it isunderstood that any amplification method that uses a primer may besuitable. Such suitable procedures include polymerase chain reaction(PCR); strand displacement amplification (SDA); nucleic acidsequence-based amplification (NASBA); cascade rolling circleamplification (CRCA), loop-mediated isothermal amplification of DNA(LAMP); isothermal and chimeric primer-initiated amplification ofnucleic acids (ICAN); target based-helicase dependant amplification(HDA); transcription-mediated amplification (TMA), and the like.Therefore, when the term PCR is used, it should be understood to includeother alternative amplification methods. It is understood that protocolsmay need to be adjusted accordingly.

FIG. 1 shows an illustrative self-contained nucleic acid analysis pouch10. Pouch 10 has a cell lysis zone 20, a nucleic acid preparation zone40, a first-stage amplification zone 60, and a second-stageamplification zone 80. A sample containing nucleic acid is introducedinto the pouch 10 via sample injection port 12. Pouch 10 comprises avariety of channels and blisters of various sizes and is arranged suchthat the sample flows through the system. The sample passes through thevarious zones and is processed accordingly.

Sample processing occurs in various blisters located within pouch 10.Various channels are provided to move the sample within and betweenprocessing zones, while other channels are provided to deliver fluidsand reagents to the sample or to remove such fluids and reagents fromthe sample. Liquid within pouch 10 illustratively is moved betweenblisters by pressure, illustratively pneumatic pressure, as describedbelow, although other methods of moving material within the pouch arecontemplated.

While other containers may be used, illustratively, pouch 10 is formedof two layers of a flexible plastic film or other flexible material suchas polyester, polyethylene terephthalate (PET), polycarbonate,polypropylene, polymethylmethacrylate, and mixtures thereof that can bemade by any process known in the art, including extrusion, plasmadeposition, and lamination. Metal foils or plastics with aluminumlamination also may be used. Other barrier materials are known in theart that can be sealed together to form the blisters and channels. Ifplastic film is used, the layers may be bonded together, illustrativelyby heat sealing. Illustratively, the material has low nucleic acidbinding capacity.

For embodiments employing fluorescent monitoring, plastic films that areadequately low in absorbance and auto-fluorescence at the operativewavelengths are preferred. Such material could be identified by tryingdifferent plastics, different plasticizers, and composite ratios, aswell as different thicknesses of the film. For plastics with aluminum orother foil lamination, the portion of the pouch that is to be read by afluorescence detection device can be left without the foil. For example,if fluorescence is monitored in the blisters 82 of the second stageamplification zone 80 of pouch 10, then one or both layers at blisters82 would be left without the foil. In the example of PCR, film laminatescomposed of polyester (Mylar, Dupont, Wilmington Del.) of about 0.0048inch (0.1219 mm) thick and polypropylene films of 0.001-0.003 inch(0.025-0.076 mm) thick perform well. Illustratively, pouch 10 is made ofa clear material capable of transmitting approximately 80%-90% ofincident light.

In the illustrative embodiment, the materials are moved between blistersby the application of pressure, illustratively pneumatic pressure, uponthe blisters and channels. Accordingly, in embodiments employingpneumatic pressure, the pouch material illustratively is flexible enoughto allow the pneumatic pressure to have the desired effect. The term“flexible” is herein used to describe a physical characteristic of thematerial of pouch. The term “flexible” is herein defined as readilydeformable by the levels of pneumatic pressure used herein withoutcracking, breaking, crazing, or the like. For example, thin plasticsheets, such as Saran™ wrap and Ziploc® bags, as well as thin metalfoil, such as aluminum foil, are flexible. However, only certain regionsof the blisters and channels need be flexible, even in embodimentsemploying pneumatic pressure. Further, only one side of the blisters andchannels need to be flexible, as long as the blisters and channels arereadily deformable. Other regions of the pouch 10 may be made of a rigidmaterial or may be reinforced with a rigid material.

Illustratively, a plastic film is used for pouch 10. A sheet of metal,illustratively aluminum, or other suitable material, may be milled orotherwise cut, to create a die having a pattern of raised surfaces. Whenfitted into a pneumatic press (illustratively A-5302-PDS, JanesvilleTool Inc., Milton Wis.), illustratively regulated at an operatingtemperature of 195° C., the pneumatic press works like a printing press,melting the sealing surfaces of plastic film only where the die contactsthe film. Various components, such as PCR primers (illustrativelyspotted onto the film and dried), antigen binding substrates, magneticbeads, and zirconium silicate beads may be sealed inside variousblisters as the pouch 10 is formed. Reagents for sample processing canbe spotted onto the film prior to sealing, either collectively orseparately. In one embodiment, nucleotide tri-phosphates (NTPs) arespotted onto the film separately from polymerase and primers,essentially eliminating activity of the polymerase until the reaction ishydrated by an aqueous sample. If the aqueous sample has been heatedprior to hydration, this creates the conditions for a true hot-start PCRand reduces or eliminates the need for expensive chemical hot-startcomponents. This separate spotting is discussed further below, withrespect to FIG. 5C, but it is understood that such spotting may be usedwith any of the embodiments discussed herein.

When pneumatic pressure is used to move materials within pouch 10, inone embodiment a “bladder” may be employed. The bladder assembly 710, aportion of which is shown in FIG. 2B, may be manufactured in a processsimilar to that of making the pouch, but individual blisters in thebladder assembly 710 include pneumatic fittings (illustratively fitting724 a) allowing individual bladders within the bladder assembly 710 tobe pressurized by a compressed gas source. Because the bladder assemblyis subjected to compressed gas and may be used multiple times, thebladder assembly may be made from tougher or thicker material than thepouch. Alternatively, bladders may be formed from a series of platesfastened together with gaskets, seals, valves, and pistons. Otherarrangements are within the scope of this invention.

When pouch 10 is placed within the instrument, the pneumatic bladderassembly 710 is pressed against one face of the pouch 10, so that if aparticular bladder is inflated, the pressure will force the liquid outof the corresponding blister in the pouch 10. In addition to pneumaticbladders corresponding to many of the blisters of pouch 10, the bladderassembly may have additional pneumatic actuators, such as bladders orpneumatically-driven pistons, corresponding to various channels of pouch10. When activated, these additional pneumatic actuators form pinchvalves to pinch off and close the corresponding channels. To confineliquid within a particular blister of pouch 10, the pinch valvepneumatic actuators are inflated over the channels leading to and fromthe blister, such that the actuators function as pinch valves to pinchthe channels shut. Illustratively, to mix two volumes of liquid indifferent blisters, the pinch valve pneumatic actuator sealing theconnecting channel is depressurized, and the pneumatic bladders over theblisters are alternately pressurized, forcing the liquid back and forththrough the channel connecting the blisters to mix the liquid therein.The pinch valve pneumatic actuators may be of various shapes and sizesand may be configured to pinch off more than one channel at a time. Suchan illustrative pinch valve is illustrated in FIG. 1 as pinch valve 16,which may be used to close all injection ports. While pneumaticactuators are discussed herein, it is understood that other ways ofproviding pressure to the pouch are contemplated, including variouselectromechanical actuators such as linear stepper motors, motor-drivencams, rigid paddles driven by pneumatic, hydraulic or electromagneticforces, rollers, rocker-arms, and in some cases, cocked springs. Inaddition, there are a variety of methods of reversibly or irreversiblyclosing channels in addition to applying pressure normal to the axis ofthe channel. These include kinking the bag across the channel,heat-sealing, rolling an actuator, and a variety of physical valvessealed into the channel such as butterfly valves and ball valves.Additionally, small Peltier devices or other temperature regulators maybe placed adjacent the channels and set at a temperature sufficient tofreeze the fluid, effectively forming a seal. Also, while the design ofFIG. 1 is adapted for an automated instrument featuring actuatorelements positioned over each of the blisters and channels, it is alsocontemplated that the actuators could remain stationary, and the pouchcould be transitioned in one or two dimensions such that a small numberof actuators could be used for several of the processing stationsincluding sample disruption, nucleic-acid capture, first andsecond-stage PCR, and other applications of the pouch such asimmunoassay and immuno-PCR. Rollers acting on channels and blisterscould prove particularly useful in a configuration in which the pouch istranslated between stations. Thus, while pneumatic actuators are used inthe presently disclosed embodiments, when the term “pneumatic actuator”is used herein, it is understood that other actuators and other ways ofproviding pressure may be used, depending on the configuration of thepouch and the instrument.

With reference to FIG. 1, an illustrative sample pouch 10 configured fornucleic acid extraction and multiplex PCR is provided. The sample enterspouch 10 via sample injection port 12 in fitment 90. Injector port 12may be a frangible seal, a one-way valve, or other entry port. Vacuumfrom inside pouch 10 may be used to draw the sample into pouch 10, asyringe or other pressure may be used to force the sample into pouch 10,or other means of introducing the sample into pouch 10 via injector port12 may be used. The sample travels via channel 14 to the three-lobedblister 22 of the cell lysis zone 20, wherein cells in the sample arelysed. Once the sample enters three-lobed blister 22, pinch valve 16 isclosed. Along with pinch valve 36, which may have been already closed,the closure of pinch valve 16 seals the sample in three-lobed blister22. It is understood that cell lysis may not be necessary with everysample. For such samples, the cell lysis zone may be omitted or thesample may be moved directly to the next zone. However, with manysamples, cell lysis is needed. In one embodiment, bead-milling is usedto lyse the cells.

Bead-milling, by shaking or vortexing the sample in the presence oflysing particles such as zirconium silicate (ZS) beads 34, is aneffective method to form a lysate. It is understood that, as usedherein, terms such as “lyse,” “lysing,” and “lysate” are not limited torupturing cells, but that such terms include disruption of non-cellularparticles, such as viruses. FIG. 2A displays one embodiment of a celllysis zone 20, where convergent flow creates high velocity bead impacts,to create lysate. Illustratively, the two lower lobes 24, 26 ofthree-lobed blister 22 are connected via channel 30, and the upper lobe28 is connected to the lower lobes 24, 26 at the opposing side 31 ofchannel 30. FIG. 2B shows a counterpart portion of the bladder assembly710 that would be in contact with the cell lysis zone 20 of the pouch10. When pouch 10 is placed in an instrument, adjacent each lobe 24, 26,28 on pouch 10 is a corresponding pneumatic bladder 724, 726, 728 in thebladder assembly 710. It is understood that the term “adjacent,” whenreferring to the relationship between a blister or channel in a pouchand its corresponding pneumatic actuator, refers to the relationshipbetween the blister or channel and the corresponding pneumatic actuatorwhen the pouch is placed into the instrument. In one embodiment, thepneumatic fittings 724 a, 726 a of the two lower pneumatic bladders 724,726 adjacent lower lobes 24, 26 are plumbed together. The pneumaticfittings 724 a, 726 a and the pneumatic fitting 728 a of upper pneumaticbladder 728 adjacent upper lobe 28 are plumbed to the opposing side ofan electrically actuated valve configured to drive a double-actingpneumatic cylinder. Thus configured, pressure is alternated between theupper pneumatic bladder 728 and the two lower pneumatic bladders 724,726. When the valve is switched back and forth, liquid in pouch 10 isdriven between the lower lobes 24, 26 and the upper lobe 28 through anarrow nexus 32 in channel 30. As the two lower lobes 24, 26 arepressurized at the same time, the flow converges and shoots into theupper lobe 28. Depending on the geometry of the lobes, the collisionvelocity of beads 34 at the nexus 32 may be at least about 12 m/sec,providing high-impact collisions resulting in lysis. The illustrativethree-lobed system allows for good cell disruption and structuralrobustness, while minimizing size and pneumatic gas consumption. WhileZS beads are used as the lysing particles, it is understood that thischoice is illustrative only, and that other materials and particles ofother shapes may be used. It is also understood that otherconfigurations for cell lysis zone 20 are within the scope of thisinvention.

While a three-lobed blister is used for cell lysis, it is understoodthat other multi-lobed configurations are within the scope of thisinvention. For instance, a four-lobed blister, illustratively in acloverleaf pattern, could be used, wherein the opposite blisters arepressurized at the same time, forcing the lysing particles toward eachother, and then angling off to the other two lobes, which then may bepressurized together. Such a four-lobed blister would have the advantageof having high-velocity impacts in both directions. Further, it iscontemplated that single-lobed blisters may be used, wherein the lysingparticles are moved rapidly from one portion of the single-lobed blisterto the other. For example, pneumatic actuators may be used to close offareas of the single-lobed blister, temporarily forming a multi-lobedblister in the remaining areas. Other actuation methods may also be usedsuch as motor, pneumatic, hydraulic, or electromagnetically-drivenpaddles acting on the lobes of the device. Rollers or rotary paddles canbe used to drive fluid together at the nexus 32 of FIG. 2A,illustratively if a recirculation means is provided between the upperand lower lobes and the actuator provides peristaltic pumping action.Other configurations are within the scope of this invention.

It may also be possible to move the sample and lysing particles quicklyenough to effect lysis within a single-lobed lysis blister withouttemporarily forming a multi-lobed blister. In one such alternativeembodiment, as shown in FIG. 2B, vortexing may be achieved by impactingthe pouch with rotating blades or paddles 21 attached to an electricmotor 19. The blades 21 may impact the pouch at the lysis blister or mayimpact the pouch near the lysis blister, illustratively at an edge 17adjacent the lysis blister. In such an embodiment, the lysis blister maycomprise one or more blisters. FIG. 12A shows an embodiment comprisingone such lysis blister 522. FIG. 12B shows a bead beating motor 19,comprising blades 21 that may be mounted on a first side 811 of secondsupport member 804, of instrument 800 shown in FIG. 8. It is understood,however, that motor 19 may be mounted on first support member 802 or onother structure of instrument 800.

FIG. 2B also shows pneumatic bladder 716 with pneumatic fitting 716 a,and pneumatic bladder 736 with pneumatic fitting 736 a. When the pouch10 is placed in contact with bladder assembly 710, bladder 716 lines upwith channel 12 to complete pinch valve 16. Similarly, bladder 736 linesup with channel 38 to complete pinch valve 36. Operation of pneumaticbladders 716 and 736 allow pinch valves 16 and 36 to be opened andclosed. While only the portion of bladder assembly 710 adjacent the celllysis zone is shown, it is understood that bladder assembly 710 would beprovided with similar arrangements of pneumatic blisters to control themovement of fluids throughout the remaining zones of pouch 10.

Other prior art instruments teach PCR within a sealed flexiblecontainer. See, e.g., U.S. Pat. Nos. 6,645,758 and 6,780,617, andco-pending U.S. patent application Ser. No. 10/478,453, hereinincorporated by reference. However, including the cell lysis within thesealed PCR vessel can improve ease of use and safety, particularly ifthe sample to be tested may contain a biohazard. In the embodimentsillustrated herein, the waste from cell lysis, as well as that from allother steps, remains within the sealed pouch. However, it is understoodthat the pouch contents could be removed for further testing.

Once the cells are lysed, pinch valve 36 is opened and the lysate ismoved through channel 38 to the nucleic acid preparation zone 40, asbest seen in FIG. 3, after which, pinch valve 36 is closed, sealing thesample in nucleic acid preparation zone 40. In the embodimentillustrated in FIG. 3, purification of nucleic acids takes thebead-milled material and uses affinity binding to silica-basedmagnetic-beads 56, washing the beads with ethanol, and eluting thenucleic acids with water or other fluid, to purify the nucleic acid fromthe cell lysate. The individual components needed for nucleic acidextraction illustratively reside in blisters 44, 46, 48, which areconnected by channels 45, 47, 49 to allow reagent mixing. The lysateenters blister 44 from channel 38. Blister 44 may be provided withmagnetic beads 56 and a suitable binding buffer, illustratively ahigh-salt buffer such as that of 1-2-3™ Sample Preparation Kit (IdahoTechnology, Salt Lake City, Utah) or either or both of these componentsmay be provided subsequently through one or more channels connected toblister 44. The nucleic acids are captured on beads 56, pinch valve 53is then opened, and the lysate and beads 56 may be mixed by gentlepressure alternately on blisters 44 and 58 and then moved to blister 58via pneumatic pressure illustratively provided by a correspondingpneumatic bladder on bladder assembly 710. The magnetic beads 56 arecaptured in blister 58 by a retractable magnet 50, which is located inthe instrument adjacent blister 58, and waste may be moved to a wastereservoir or may be returned to blister 44 by applying pressure toblister 58. Pinch valve 53 is then closed. The magnetic beads 56 arewashed with ethanol, isopropanol, or other organic or inorganic washsolution provided from blister 46, upon release of pinch valve 55.Optionally, magnet 50 may be retracted allowing the beads to be washedby providing alternate pressure on blisters 46 and 58. The beads 56 areonce again captured in blister 58 by magnet 50, and the non-nucleic acidportion of the lysate is washed from the beads 56 and may be moved backto blister 46 and secured by pinch valve 55 or may be washed away viaanother channel to a waste reservoir. Once the magnetic beads arewashed, pinch valve 57 is opened, releasing water (illustrativelybuffered water) or another nucleic acid eluant from blister 48. Onceagain, the magnet 50 may be retracted to allow maximum mixing of waterand beads 56, illustratively by providing alternate pressure on blisters48 and 58. The magnet 50 is once again deployed to collect beads 56.Pinch valve 59 is released and the eluted nucleic acid is moved viachannel 52 to first-stage amplification zone 60. Pinch valve 59 is thenclosed, thus securing the sample in first-stage amplification zone 60.

It is understood that the configuration for the nucleic acid preparationzone 40, as shown in FIG. 3 and described above, is illustrative only,and that various other configurations are possible within the scope ofthe present disclosure.

The ethanol, water, and other fluids used herein may be provided to theblisters in various ways. The fluids may be stored in the blisters, thenecks of which may be pinched off by various pinch valves or frangibleportions that may be opened at the proper time in the sample preparationsequence. Alternatively, fluid may be stored in reservoirs in the pouchas shown pouch 110 in FIG. 5A, or in the fitment as discussed withrespect to pouch 210 of FIG. 6A, and moved via channels, as necessary.In still another embodiment, the fluids may be introduced from anexternal source, as shown in FIG. 1, especially with respect to ethanolinjection ports 41, 88 and plungers 67, 68, 69. Illustratively, plungers67, 68, 69 may inserted into fitment 90, illustratively of a more rigidmaterial, and may provide a measured volume of fluid upon activation ofthe plunger, as in U.S. patent application Ser. No. 10/512,255, hereinincorporated by reference. The measured volume may be the same ordifferent for each of the plungers. Finally, in yet another embodiment,the pouch may be provided with a measured volume of the fluid that isstored in one or more blisters, wherein the fluid is contained withinthe blister, illustratively provided in a small sealed pouch within theblister, effectively forming a blister within the blister. At theappropriate time, the sealed pouch may then be ruptured, illustrativelyby pneumatic pressure, thereby releasing the fluid into the blister ofthe pouch. The instrument may also be configured the provide some or allof the reagents directly through liquid contacts between the instrumentand the fitment or pouch material provided that the passage of fluid istightly regulated by a one-way valve to prevent the instrument frombecoming contaminated during a run. Further, it will often be desirablefor the pouch or its fitment to be sealed after operation to prohibitcontaminating DNA to escape from the pouch. Various means are known toprovide reagents on demand such as syringe pumps, and to make temporaryfluid contact with the fitment or pouch, such as barbed fittings oro-ring seals. It is understood that any of these methods of introducingfluids to the appropriate blister may be used with any of theembodiments of the pouch as discussed herein, as may be dictated by theneeds of a particular application.

As discussed above, nested PCR involves target amplification performedin two stages. In the first-stage, targets are amplified, illustrativelyfrom genomic or reverse-transcribed template. The first-stageamplification may be terminated prior to plateau phase, if desired. Inthe secondary reaction, the first-stage amplicons may be diluted and asecondary amplification uses the same primers or illustratively usesnested primers hybridizing internally to the primers of the first-stageproduct. Nested primers may be fully internal to the first-stageamplification primers, or there may be some overlap with the nestedprimers shifted in the 3′-direction. Advantages of nested PCR include:a) the initial reaction product forms a homogeneous and specifictemplate assuring high fidelity in the secondary reaction, wherein evena relatively low-efficiency first-stage reaction creates adequatetemplate to support robust second-stage reaction; b) nonspecificproducts from the first-stage reaction do not significantly interferewith the second stage reaction, as different nested primers are used andthe original amplification template (illustratively genomic DNA orreverse-transcription product) may be diluted to a degree thateliminates its significance in the secondary amplification; and c)nested PCR enables higher-order reaction multiplexing. First-stagereactions can include primers for several unique amplification products.These products are then identified in the second-stage reactions.However, it is understood that first-stage multiplex and second-stagesingleplex is illustrative only and that other configurations arepossible. For example, the first-stage may amplify a variety ofdifferent related amplicons using a single pair of primers, andsecond-stage may be used to target differences between the amplicons,illustratively using melting curve analysis.

Turning back to FIG. 1, the nucleic acid sample enters the first-stageamplification zone 60 via channel 52 and is delivered to blister 61. APCR mixture, including a polymerase (illustratively a Taq polymerase),dNTPs, and primers, illustratively a plurality of pairs of primers formultiplex amplification, may be provided in blister 61 or may beintroduced into blister 61 via various means, as discussed above.Alternatively, dried reagents may be spotted onto the location ofblister 61 upon assembly of pouch 10, and water or buffer may beintroduced to blister 61, illustratively via plunger 68, as shown inFIG. 1. As best seen in FIG. 4, the sample is now secured in blister 61by pinch valves 59 and 72, and is thermocycled between two or moretemperatures, illustratively by heat blocks or Peltier devices that arelocated in the instrument and configured to contact blister 61. However,it is understood that other means of heating and cooling the samplecontained within blister 61, as are known in the art, are within thescope of this invention. Non-limiting examples of alternativeheating/cooling devices for thermal cycling include having a air-cycledblister within the bladder, in which the air in the pneumatic blisteradjacent blister 61 is cycled between two or more temperatures; ormoving the sample to temperature zones within the blister 61,illustratively using a plurality of pneumatic presses, as in U.S. patentapplication Ser. No. 10/478,453, herein incorporated by reference, or bytranslating pouch 10 on an axis or providing pouch 10 with a rotarylayout and spinning pouch 10 to move the contents between heat zones offixed temperature.

Nucleic acids from pathogens are often co-isolated with considerablequantities of host nucleic acids. These host-derived nucleic acids ofteninteract with primers, resulting in amplification of undesired productsthat then scavenge primers, dNTPs, and polymerase activity, potentiallystarving a desired product of resources. Nucleic acids from pathogenicorganisms are generally of low abundance, and undesired product is apotential problem. The number of cycles in the first-stage reaction ofzone 60 may be optimized to maximize specific products and minimizenon-specific products. It is expected that the optimum number of cycleswill be between about 10 to about 30 cycles, illustratively betweenabout 15 to about 20 cycles, but it is understood that the number ofcycles may vary depending on the particular target, host, and primersequence.

Following the first-stage multiplex amplification, the first-stageamplification product is diluted, illustratively in incomplete PCRmaster mix, before fluidic transfer to secondary reaction sites.

FIG. 4 shows an illustrative embodiment for diluting the sample in threesteps. In the first step, pinch valve 72 is opened and the sampleundergoes a two-fold dilution by mixing the sample in blister 61 with anequal volume of water or buffer from blister 62, which is provided toblister 62, as well as blisters 64 and 66, as discussed above. Squeezingthe volume back and forth between blisters 61, 62 provides thoroughmixing. As above, mixing may be provided by pneumatic bladders providedin the bladder 710 and located adjacent blisters 61, 62. The pneumaticbladders may be alternately pressurized, forcing the liquid back andforth. During mixing, a pinch valve 74 prevents the flow of liquid intothe adjacent blisters. At the conclusion of mixing, a volume of thediluted sample is captured in region 70, and pinch valve 72 is closed,sealing the diluted sample in region 70. Pinch valve 74 is opened andthe sample is further diluted by water or buffer provided in either orboth of blisters 63, 64. As above, squeezing the volume back and forthbetween blisters 63, 64 provides mixing. Subsequently, pinch valve 74 isclosed, sealing a further diluted volume of sample in region 71. Finaldilution takes place illustratively by using buffer or water provided ineither or both of blisters 65, 66, with mixing as above. Illustrativelythis final dilution takes place using an incomplete PCR master mix(e.g., containing all PCR reagents except primers) as the fluid.Optional heating of the contents of blister 66 prior to second-stageamplification can provide the benefits of hot-start amplificationwithout the need for expensive antibodies or enzymes. It is understood,however, that water or other buffer may be used for the final dilution,with additional PCR components provided in second-stage amplificationzone 80. While the illustrative embodiment uses three dilution stages,it is understood that any number of dilution stages may be used, toprovide a suitable level of dilution. It is also understood that theamount of dilution can be controlled by adjusting the volume of thesample captured in regions 70 and 71, wherein the smaller the amount ofsample captured in regions 70 and 71, the greater the amount of dilutionor wherein additional aliquots captured in region 70 and/or region 71 byrepeatedly opening and closing pinch valves 72 and 74 and/or pinchvalves 74 and 76 may be used to decrease the amount of dilution. It isexpected that about 10⁻² to about 10⁻⁵ dilution would be suitable formany applications.

Success of the secondary PCR reactions is dependent upon templategenerated by the multiplex first-stage reaction. Typically, PCR isperformed using DNA of high purity. Methods such as phenol extraction orcommercial DNA extraction kits provide DNA of high purity. Samplesprocessed through the pouch 10 may require accommodations be made tocompensate for a less pure preparation. PCR may be inhibited bycomponents of biological samples, which is a potential obstacle.Illustratively, hot-start PCR, higher concentration of taq polymeraseenzyme, adjustments in MgCl₂ concentration, adjustments in primerconcentration, and addition of adjuvants (such as DMSO, TMSO, orglycerol) optionally may be used to compensate for lower nucleic acidpurity. While purity issues are likely to be more of a concern withfirst-stage amplification, it is understood that similar adjustments maybe provided in the second-stage amplification as well.

While dilution and second-stage sample preparation are accomplished inthe illustrative embodiment by retaining a small amount of amplifiedsample in the blisters and channels of the first-stage PCR portion ofthe pouch, it is understood that these processes may also be performedin other ways. In one such illustrative example, pre-amplified samplecan be captured in a small cavity in a member, illustratively atranslating or rotating member, able to move a fixed volume of samplefrom the first to the second-stage PCR reagent. A one microliterfraction of the pre-amplified sample, mixed with 100 microliters offresh PCR reagent would yield a one-hundred-fold reduction inconcentration. It is understood that this dilution is illustrative only,and that other volumes and dilution levels are possible. This approachcould be accomplished by forcing the first-stage amplification productinto the rigid fitment where it contacts one of the plungers 68 or 69 ofFIG. 1. In such an embodiment, the plunger would be configured to carrya small fraction of the sample into contact with the adjacent dilutionbuffer or second-stage PCR buffer. Similarly a sliding element could beused to carry a small amount of the first-stage amplification productinto contact with the second-stage reaction mix while maintaining a sealbetween the stages, and containing the amplified sample within the rigidfitment 90.

Subsequent to first-stage PCR and dilution, channel 78 transfers thesample to a plurality of low volume blisters 82 for secondary nestedPCR. In one illustrative embodiment, dried primers provided in thesecond-stage blisters are resuspended by the incoming aqueous materialto complete the reaction mixture. Optionally, fluorescent dyes such asLCGreen® Plus (Idaho Technology, Salt Lake City, Utah) used fordetection of double-stranded nucleic acid may be provided in eachblister or may be added to the incomplete PCR master mix provided at theend of the serial dilution, although it is understood that LCGreen® Plusis illustrative only and that other dyes are available, as are known inthe art. In another optional embodiment, dried fluorescently labeledoligonucleotide probes configured for each specific amplicon may beprovided in each respective second-stage blister, along with therespective dried primers. Further, while pouch 10 is designed to containall reactions and manipulations within, to reduce contamination, in somecircumstances it may be desirable to remove the amplification productsfrom each blister 82 to do further analysis. Other means for detectionof the second-stage amplicon, as are known in the art, are within thescope of this invention. Once the sample is transferred to blisters 82,pinch valves 84 and 86 are activated to close off blisters 82. Eachblister 82 now contains all reagents needed for amplification of aparticular target. Illustratively, each blister may contain a uniquepair of primers, or a plurality of blisters 82 may contain the sameprimers to provide a number of replicate amplifications.

It is noted that the embodiments disclosed herein use blisters for thesecond-stage amplification, wherein the blisters are formed of the sameor similar plastic film as the rest of the flexible portion. However, inmany embodiments, the contents of the second-stage blisters are neverremoved from the second-stage blisters, and, therefore, there is no needfor the second-stage reaction to take place in flexible blisters. It isunderstood that the second-stage reaction may take place in a pluralityof rigid, semi-rigid, or flexible chambers that are fluidly connected tothe blisters. The chambers could be sealed as in the present example byplacing pressure on flexible channels that connect the chambers, or maybe sealed in other ways, illustratively by heat sealing or use ofone-way valves. Various embodiments discussed herein include blistersprovided solely for the collection of waste. Since the waste may neverbe removed, waste could be collected in rigid, semi-rigid, or flexiblechambers.

It is within the scope of this invention to do the second-stageamplification with the same primers used in the first-stageamplification (see U.S. Pat. No. 6,605,451). However, it is oftenadvantageous to have primers in second-stage reactions that are internalto the first-stage product such that there is no or minimal overlapbetween the first- and second-stage primer binding sites. Dilution offirst-stage product largely eliminates contribution of the originaltemplate DNA and first-stage reagents to the second-stage reaction.Furthermore, illustratively, second-stage primers with a Tm higher thanthose used in the first-stage may be used to potentiate nestedamplification. Primers may be designed to avoid significant hairpins,hetero/homo-dimers and undesired hybridization. Because of the nestedformat, second-stage primers tolerate deleterious interactions far moreso than primers used to amplify targets from genomic DNA in a singlestep. Optionally, hot-start is used on second-stage amplification.

If a fluorescent dye is included in second-stage amplification,illustratively as a dsDNA binding dye or as part of a fluorescent probe,as are known in the art, optics provided may be used to monitoramplification of one or more of the samples. Optionally, analysis of theshape of the amplification curve may be provided to call each samplepositive or negative. Illustrative methods of calling the sample arediscussed in U.S. Pat. No. 6,730,501, herein incorporated by reference.Alternatively, methods employing a crossing threshold may be used. Acomputer may be provided externally or within the instrument and may beconfigured to perform the methods and call the sample positive ornegative based upon the presence or absence of second-stageamplification, and may provide quantitative information about thestarting template concentration by comparing characteristic parametersof the amplification curve (such as crossing threshold) to standardcurves, or relative to other amplification curves within the run. It isunderstood, however, that other methods, as are known in the art, may beused to call each sample. Other analyses may be performed on thefluorescent information. One such non-limiting example is the use ofmelting curve analysis to show proper melting characteristics (e.g. Tm,melt profile shape) of the amplicon. The optics provided may beconfigured to capture images of all blisters 82 at once, or individualoptics may be provided for each individual blister. Other configurationsare within the scope of this invention.

FIG. 5A shows an alternative pouch 110. In this embodiment, variousreagents are loaded into pouch 110 via fitment 190. FIG. 5B shows across-section of fitment 190 with one of a plurality of plungers 168. Itis understood that, while FIG. 5B shows a cross-section through entrychannel 115 a, as shown in the embodiment of FIG. 5A, there are 12 entrychannels present (entry channel 115 a through 115 l), each of which mayhave its own plunger 168 for use in fitment 190, although in thisparticular configuration, entry channels 115 c, 115 f, and 115 i are notused. It is understood that a configuration having 12 entry channels isillustrative only, and that any number of entry channels and associatedplungers may be used. In the illustrative embodiment, an optional vacuumport 142 of fitment 190 is formed through a first surface 194 of fitment190 to communicate with chamber 192. Optional vacuum port 142 may beprovided for communication with a vacuum or vacuum chamber (not shown)to draw out the air from within pouch 110 to create a vacuum withinchamber 192 and the various blisters and chambers of pouch 110. Plunger168 is then inserted far enough into chamber 192 to seal off vacuum port142. Chamber 192 is illustratively provided under a predetermined amountof vacuum to draw a desired volume of liquid into chamber 192 upon use.Additional information on preparing chamber 192 may be found in U.S.patent application Ser. No. 10/512,255, already incorporated byreference.

Illustrative fitment 19Q further includes an injection port 141 formedin the second surface 195 of fitment 190. Illustratively, injection port141 is positioned closer to the plastic film portion of pouch 110 thanvacuum port 142, as shown in FIG. 5B, such that the plunger 168 isinserted far enough to seal off vacuum port 142, while still allowingaccess to chamber 192 via injection port 141. As shown, second surface119 of plastic film portion 117 provides a penetrable seal 139 toprevent communication between chamber 192 and the surrounding atmospherevia injection port 141. However, it is understood that second surface119 optionally may not extend to injection port 141 and various otherseals may be employed. Further, if another location for the seal isdesired, for example on a first surface 194 of fitment 190, injectionport 141 may include a channel to that location on fitment 190. U.S.patent application Ser. No. 10/512,255, already incorporated byreference, shows various configurations where the seal is locatedremotely from the injection port, and the seal is connected to thechamber via a channel. Also, U.S. patent application Ser. No. 10/512,255discloses various configurations where channels connect a single seal tomultiple chambers. Variations in seal location, as well as connection ofa single injection port to multiple chambers, are within the scope ofthis invention. Optionally, seal 139 may be frangible and may be brokenupon insertion of a cannula (not shown), to allow a fluid sample fromwithin the cannula to be drawn into or forced into chamber 192.

The illustrative plunger 168 of the pouch assembly 110 is cylindrical inshape and has a diameter of approximately 5 mm to be press-fit intochamber 192. Plunger 168 includes a first end portion 173 and anopposite second end portion 175. As shown in FIG. 5B, a notch 169 ofplunger 168 is formed in second end portion 175. In use, second endportion 175 is inserted part way into chamber 192, and notch 169 may bealigned with injection port 141 to allow a fluid sample to be drawn intoor injected into chamber 192, even when plunger 168 is inserted farenough that plunger 168 would otherwise be blocking injection port 141.

Illustratively, a fluid is placed in a container (not shown) with asyringe having a cannulated tip that can be inserted into injection port141 to puncture seal 139 therein. In using an air-evacuated pouchassembly 110, when seal 139 is punctured, the fluid is withdrawn fromthe container due to the negative pressure within chamber 192 relativeto ambient air pressure. Fluid then passes through port 141 to fillchamber 192. At this point, the fluid usually does not flow into theplastic film portion 117 of pouch 110. Finally, the plunger 168 isinserted into chamber 192 such that second end portion 175 of plunger168 approaches the bottom 191 of chamber 192, to push a measured amountof the reagent or sample into the plastic film portion 117. As shown,plunger 168 is configured such that upon full insertion, second endportion 175 does not quite meet bottom 191 of chamber 192. The remainingspace is useful in trapping bubbles, thereby reducing the number ofbubbles entering plastic film portion 117. However, in some embodimentsit may be desirable for second end portion 175 to meet bottom 191 uponfull insertion of plunger 168. In the embodiment shown in FIG. 5A, entrychannels 115 a, 115 b, 115 d, 115 e, 115 g, 115 h, 115 j, 115 k, and 115l all lead to reaction zones or reservoir blisters. It is understoodthat full insertion of the plunger associated with entry channel 115 awould force a sample into three-lobed blister 122, full insertion of theplunger associated with entry channel 115 b would force a reagent intoreservoir blister 101, full insertion of the plunger associated withentry channel 115 d would force a reagent into reservoir blister 102,full insertion of the plunger associated with entry channel 115 e wouldforce a reagent into reservoir blister 103, full insertion of theplunger associated with entry channel 115 g would force a reagent intoreservoir blister 104, full insertion of the plunger associated withentry channel 115 h would force a reagent into reservoir blister 105,full insertion of the plunger associated with entry channel 115 j wouldforce a reagent into reservoir blister 106, full insertion of theplunger associated with entry channel 115 k would force a reagent intoreservoir blister 107, and full insertion of the plunger associated withentry channel 115 l would force a reagent into reservoir blister 108.

If a plunger design is used including notch 169 as illustrated in theembodiment shown in FIG. 5B, the plunger 168 may be rotated prior tobeing lowered, so as to offset notch 169 and to close off injection port141 from communication with chamber 192, to seal the contents therein.This acts to minimize any potential backflow of fluid through injectionport 141 to the surrounding atmosphere, which is particularly usefulwhen it is desired to delay in full insertion of the plunger. Althoughnotch 169 is shown and described above with respect to plunger 168, itis within the scope of this disclosure to close off injection port 141soon after dispensing the fluid sample into the chamber 192 by othermeans, such as depressing plunger 168 toward the bottom of chamber 192,heat sealing, unidirectional valves, or self-sealing ports, for example.If heat sealing is used as the sealing method, a seal bar could beincluded in the instrument such that all chambers are heat sealed uponinsertion of the pouch into the instrument.

In the illustrative method, the user injects the sample into theinjection port 141 associated with entry channel 115 a, and water intothe various other injection ports. The water rehydrates reagents thathave been previously freeze-dried into chambers 192 associated with eachof entry channels 115 b, 115 d, 115 e, 115 g, 115 h, 115 j, 115 k, and115 l. The water may be injected through one single seal and then bedistributed via a channel to each of the chambers, as shown in FIG. 6Abelow, or the water could be injected into each chamber independently.Alternatively, rather than injecting water to rehydrate dried reagents,wet reagents such as lysis reagents, nucleic acid extraction reagents,and PCR reagents may be injected into the appropriate chambers 192 ofthe fitment 190.

Upon activation of the plunger 168 associated with entry channel 115 a,the sample is forced directly into three-lobed blister 122 via channel114. The user also presses the remaining plungers 168, forcing thecontents out of each of the chambers 192 in fitment 190 and intoreservoir blisters 101 through 108. At this point, pouch 110 is loadedinto an instrument for processing. While instrument 800, shown in FIG.8, is configured for the pouch 210 of FIG. 6A, it is understood thatmodification of the configuration of the bladders of instrument 800would render instrument 800 suitable for use with pouches 110 and 510,or with pouches of other configurations.

In one illustrative example, upon depression of the plungers 168,reservoir blister 101 now contains DNA-binding magnetic beads inisopropanol, reservoir blister 102 now contains a first wash solution,reservoir blister 103 now contains a second wash solution, reservoirblister 104 now contains a nucleic acid elution buffer, reservoirblister 105 now contains first-stage PCR reagents, including multiplexedfirst-stage primers, reservoir blister 106 now contains second-stage PCRreagents without primers, reservoir blister 107 now contains negativecontrol PCR reagents without primers and without template, and reservoirblister 108 now contains positive control PCR reagents with template.However, it is understood that these reagents are illustrative only, andthat other reagents may be used, depending upon the desired reactionsand optimization conditions.

Once pouch 110 has been placed into instrument 800 and the sample hasbeen moved to three-lobed blister 122, the sample may be subjected todisruption by agitating the sample with lysing particles such as ZS orceramic beads. The lysing particles may be provided in three-lobedblister 122, or may be injected into three-lobed blister 122 along withthe sample. The three-lobed blister 122 of FIG. 5A is operated in muchthe same way as three-lobed blister 22 of FIG. 1, with the two lowerlobes 124, 126 pressurized together, and pressure is alternated betweenthe upper lobe 128 and the two lower lobes 124, 126. However, asillustrated, lower lobes 124, 126 are much more rounded than lower lobes24, 26, allowing for a smooth flow of beads to channel 130 and allowingfor high-speed collisions, even without the triangular flow separator atnexus 32. As with three-lobed blister 22, three-lobed blister 122 ofFIG. 5A allows for effective lysis or disruption of microorganisms,cells, and viral particles in the sample. It has been found that achannel 130 having a width of about 3-4 mm, and illustratively about 3.5mm, remains relatively clear of beads during lysis and is effective inproviding for high-velocity collisions.

After lysis, nucleic-acid-binding magnetic beads are injected into upperlobe 128 via channel 138 by pressurizing a bladder positioned overreservoir blister 101. The magnetic beads are mixed, illustratively moregently than with during lysis, with the contents of three-lobed blister122, and the solution is incubated, illustratively for about 1 minute,to allow nucleic acids to bind to the beads.

The solution is then pumped into the “FIG. 8” blister 144 via channel143, where the beads are captured by a retractable magnet housed in theinstrument, which is illustratively pneumatically driven. The beadcapture process begins by pressurizing all lobes 124, 126, and 128 ofthe bead milling apparatus 122. This forces much of the liquid contentsof 122 through channel 143 and into blister 144. A magnet is broughtinto contact with the lower portion 144 b of blister 144 and the sampleis incubated for several seconds to allow the magnet to capture thebeads from the solution, then the bladders adjacent to blister 122 aredepressurized, the bladders adjacent blister portions 144 a and 144 bare pressurized, and the liquid is forced back into blister 122. Sincenot all of the beads are captured in a single pass, this process may berepeated up to 10 times to capture substantially all of the beads inblister 144. Then the liquid is forced out of blister 144, leavingbehind only the magnetic beads and the captured nucleic acids, and washreagents are introduced into blister 144 in two successive washes (fromreservoir blisters 102 and 103 via channels 145 and 147, respectively).In each wash, the bladder positioned over the reservoir blistercontaining the wash reagent is pressurized, forcing the contents intoblister 144. The magnet is withdrawn and the pellet containing themagnetic beads is disrupted by alternatively pressurizing each of twobladders covering each lobe 144 a and 144 b of blister 144. When theupper lobe 144 a is compressed, the liquid contents are forced into thelower lobe 144 b, and when the lower lobe 144 b is compressed, thecontents are forced into the upper lobe 144 a. By agitating the solutionin blister 144 between upper lobe 144 a and lower lobe 144 b, themagnetic beads are effectively washed of impurities. A balance ismaintained between inadequate agitation, leaving the pellet of beadsundisturbed, and excessive agitation, potentially washing the nucleicacids from the surface of the beads and losing them with the washreagents. After each wash cycle, the magnetic beads are captured via themagnet in blister 144 and the wash reagents are illustratively forcedinto three-lobed blister 122, which now serves as a waste receptacle.However, it is understood that the used reservoir blisters may alsoserve as waste receptacles, or other blisters may be providedspecifically as waste receptacles.

Nucleic acid elution buffer from reservoir blister 104 is then injectedvia channel 149 into blister 144, the sample is once again agitated, andthe magnetic beads are recaptured by employment of the magnet. The fluidmixture in blister 144 now contains nucleic acids from the originalsample. Pressure on blister 144 moves the nucleic acid sample to thefirst stage PCR blister 161 via channel 152, where the sample is mixedwith first-stage PCR master mix containing multiple primer sets, the PCRmaster mix provided from reservoir blister 105 via channel 162. Ifdesired, the sample and/or the first-stage PCR master mix may be heatedprior to mixing, to provide advantages of hot start. Optionally,components for reverse transcription of RNA targets may be providedprior to first-stage PCR. Alternatively, an RT enzyme, illustratively athermostable RT enzyme may be provided in the first-stage PCR master mixto allow for contemporaneous reverse transcription of RNA targets. It isunderstood that an RT enzyme may be present in the first-stage PCRmixture in any of the embodiments disclosed herein. As will be seenbelow, pouch 110 of FIG. 5A is configured for up to 10 primer sets, butit is understood that the configuration may be altered and any number ofprimer sets may be used. A bladder positioned over blister 161 ispressurized at low pressure, to force the contents of blister 161 intointimate contact with a heating/cooling element, illustratively aPeltier element, on the other side of blister 161. The pressure onblister 161 should be sufficient to assure good contact with theheating/cooling element, but should be gentle enough such that fluid isnot forced from blister 161. The heating/cooling element is temperaturecycled, illustratively between about 60° C. to about 95° C.Illustratively, temperature cycling is performed for about 15-20 cycles,resulting in amplification of one or more nucleic acid targets present.Also illustratively, temperature cycling ceases prior to plateau phase,and may cease in log phase or even prior to log phase. In one example,it may be desirable merely to enrich the sample with the desiredamplicons, without reaching minimal levels of detection. See U.S. Pat.No. 6,605,451, herein incorporated by reference.

The amplified sample is optionally then diluted by forcing most thesample back into blister 144 via channel 152, leaving only a smallamount (illustratively about 1 to 5%) of the amplified sample in blister161, and second-stage PCR master mix is provided from reservoir blister106 via channel 163. The sample is thoroughly mixed illustratively bymoving it back and forth between blisters 106 and 161 via channel 163.If desired, the reaction mixture may be heated to above extensiontemperature, illustratively at least 60° C., prior to second-stageamplification. The sample is then forced through channel 165 into anarray of low volume blisters 182 in the center of second-stageamplification zone 180. Each of the ten illustrative low volume blisters182 may contain a different primer pair, either essentially the same asone of the primer pairs in the first-stage amplification, or “nested”within the first-stage primer pair to amplify a shortened amplicon. Theprimers, now hydrated by the sample, complete the amplification mixture.Positive and negative control samples are also introduced bypressurizing the contents of reservoir blisters 107 and 108,respectively, forcing PCR master mix either without target DNA fromreservoir blister 107 via channel 166, or with control DNA fromreservoir blister 108, via channel 167. As illustrated, there are fiveeach of positive control blisters 183 and negative control blisters 181,which may be multiplexed 2-fold to provide the necessary controls forten different second-stage amplification reactions. It is understoodthat this configuration is illustrative only and that any number ofsecond-stage blisters may be provided.

Illustratively, the PCR master mix used for second-stage amplificationlacks the primers, but is otherwise complete. However, an “incomplete”PCR master mix may lack other PCR components as well. In one example,the second-stage PCR master mix is water or buffer only, which is thenmixed with the optionally diluted first-stage PCR amplification product.This mixture is moved to the small-volume PCR reaction blisters, whereall of the remaining components have been previously provided. Ifdesired, all of the remaining components may be mixed together andspotted as a single mixture into the small-volume PCR reaction blisters.Alternatively, as illustrated in FIG. 5C, each of the components may bespotted onto a separate region of the small-volume PCR reaction blister182. As shown in FIG. 5C, four regions are present, illustratively withdNTPs spotted at region 182 a, primers spotted at 182 b, polymerasespotted at 182 c, and a magnesium compound spotted at 182 d. By spottingthe components separately and heating the sample mixture prior torehydrating the components, nonspecific reactions can be minimized. Itis understood that any combination of components can be spotted thisway, and that this method of spotting components into one or moreregions of the blisters may be used with any embodiment of the presentinvention.

The channels 165, 166, and 167 leading to the small-volume PCR reactionblisters 181, 182, and 183 are sealed, and a pneumatic bladder gentlypresses the array against a heating/cooling element, illustratively aPeltier element, for thermal cycling. The cycling parameters may beindependently set for second-stage thermal cycling. Illustratively, thereactions are monitored by focusing an excitation source, illustrativelya blue light (450-490 nm), onto the array, and imaging the resultantfluorescent emissions, illustratively fluorescent emissions above 510nm.

In the above example, pinch valves are not discussed. However, it isunderstood that when it is desired to contain a sample in one of theblisters, pneumatic actuators positioned over channels leading to andfrom the particular blister are pressurized, creating pinch valves andclosing off the channels. Conversely, when it is desired to move asample from one of the blisters, the appropriate pneumatic actuator isdepressurized, allowing the sample flow through the channel.

The pouch described above in FIG. 5A includes reagent reservoir blisters101 through 108, in which the user injected reagents from the fitment190 into the reagent reservoir blisters 101 through 108 in the plasticfilm portion 117 of the pouch 110, illustratively prior to insertion ofpouch 110 into the instrument. While there are advantages to the use ofthe reagent reservoir blisters of FIG. 5A, including having the abilityto maintain the contents of the various blisters at differenttemperatures, there are some disadvantages as well. Because the operatoris responsible for moving the reagents from the fitment 190 to thereservoir blisters 101 through 108, and because this is often doneoutside of the machine and thus without activated pinch valves, reagentscould occasionally leak from the reservoir blisters to the workingblisters. The reagents in reservoir blisters are exposed duringpreparation and loading. If they are pressed, squeezed, or even lightlybumped, the reagents may leak through available channels. If the loss ofreagents is substantial, the reaction may fail completely. Furthermore,during operation there may be some variability in the amount of reagentforced from the reservoir blisters 101 through 108, leading toinconsistent results. Automation of introduction of the reagents tofitment 190 and movement of the reagents from fitment 190 to reagentreservoir blisters 101 through 108 would solve many of these problems,and is within the scope of this invention.

The pouch 210 of FIG. 6A addresses many of these issues in a differentway, by using a direct-injection approach wherein the instrument itselfmoves the plungers 268, illustratively via pneumatic pistons, and forcesthe reagents into the various working blisters as the reagents areneeded. Rather than storing the reagents in reservoir blisters 101through 108 of FIG. 5A, in the embodiment of FIG. 6A the reagents areintroduced into various chambers 292 of fitment 290 and are maintainedthere until needed. Pneumatic operation of piston 268 at the appropriatetime introduces a measured amount of the reagent to the appropriatereaction blister. In addition to addressing many of the above-mentionedissues, pouch 210 also has a much more compact shape, allowing for asmaller instrument design, and pouch 210 has shorter channels,permitting better fluid flow and minimizing reagent loss in channels.

In one illustrative embodiment of FIG. 6A, a 300 μl mixture comprisingthe sample to be tested (100 μl) and lysis buffer (200 μl) is injectedinto injection port 241 a. Water is also injected into the fitment 290via seal 239 b, hydrating up to eleven different reagents, each of whichwere previously provided in dry form in chambers 292 b through 292 l viachannel 293 (shown in shadow). These reagents illustratively may includefreeze-dried PCR reagents, DNA extraction reagents, wash solutions,immunoassay reagents, or other chemical entities. For the example ofFIG. 6A, the reagents are for nucleic acid extraction, first-stagemultiplex PCR, dilution of the multiplex reaction, and preparation ofsecond-stage PCR reagents, and control reactions. In the embodimentshown in FIG. 6A, all that need be injected is the sample in port 241 aand water in port 241 b.

As shown in FIG. 6A, water injected via seal 293 b is distributed tovarious chambers via channel 293. In this embodiment, only the sampleand water need be injected into pouch 210. It is understood, however,that water could be injected into each chamber 292 independently.Further, it is understood that, rather than providing dried reagents inthe various chambers 292 and hydrating upon injection of the water,specific wet reagents could be injected into each chamber, as desired.Additionally, it is understood that one or more of chambers 292 could beprovided with water only, and the necessary reagents may be provideddried in the appropriate reaction blisters. Various combinations of theabove, as dictated by the needs of the particular reaction, are withinthe scope of this invention.

As seen in FIG. 6A, optional protrusions 213 are provided on bottomsurface 297 of fitment 290. As shown, protrusions 213 are located withintheir respective entry channels 215. However, other configurations arepossible. Protrusions 213 assist in opening entry channel 215 andprevent bottom surface 297 from engaging another flat surface in such away to pinch off entry channels 215 when plungers 268 are depressed,which helps prevent back-flow upon activation of the plungers 268. Suchprotrusions may be used on any of the various pouches according to thepresent invention.

In embodiments wherein water is injected into the pouch to hydratemultiple dry reagents in multiple chambers in the fitment, a means ofclosing the channel between the injection port and the many chambers isdesired. If the channel is not closed, activation of each plunger mayforce some of the contents of its respective chamber back out into thechannel, potentially contaminating neighboring chambers and altering thevolumes contained in and delivered from the chamber. Several ways ofclosing this channel have been used, including rotating a notchedplunger 268 as discussed above, and heat-sealing the plastic film acrossthe channel thereby closing the channel permanently, and applyingpressure to the channel as a pinch valve. Other closures may also beused, such as valves built into the fitment, illustratively one-wayvalves.

After the fluids are loaded into chambers 292 and pouch 210 is loadedinto the instrument, plunger 268 a is depressed illustratively viaactivation of a pneumatic piston, forcing the balance of the sample intothree-lobed blister 220 via channel 214. As with the embodiments shownin FIGS. 1 and 5, the lobes 224, 226, and 228 of three-lobed blister 220are sequentially compressed via action bladders 824, 826, and 828 ofbladder assembly 810, shown in FIGS. 7-9, forcing the liquid through thenarrow nexus 232 between the lobes, and driving high velocitycollisions, shearing the sample and liberating nucleic acids,illustratively including nucleic acids from hard-to-open spores,bacteria, and fungi. Cell lysis continues for an appropriate length oftime, illustratively 0.5 to 10 minutes.

Once the cells have been adequately lysed, plunger 268 b is activatedand nucleic acid binding magnetic beads stored in chamber 292 b areinjected via channel 236 into upper lobe 228 of three-lobed blister 220.The sample is mixed with the magnetic beads and the mixture is allowedto incubate for an appropriate length of time, illustrativelyapproximately 10 seconds to 10 minutes.

The mixture of sample and beads are forced through channel 238 intoblister 244 via action of bladder 826, then through channel 243 and intoblister 246 via action of bladder 844, where a retractable magnet 850located in instrument 800 adjacent blister 245, shown in FIG. 8,captures the magnetic beads from the solution, forming a pellet againstthe interior surface of blister 246. A pneumatic bladder 846, positionedover blister 246 then forces the liquid out of blister 246 and backthrough blister 244 and into blister 222, which is now used as a wastereceptacle. However, as discussed above with respect to FIG. 5A, otherwaste receptacles are within the scope of this invention. One ofplungers 268 c, 268 d, and 268 e may be activated to provide a washsolution to blister 244 via channel 245, and then to blister 246 viachannel 243. Optionally, the magnet 850 is retracted and the magneticbeads are washed by moving the beads back and forth from blisters 244and 246 via channel 243, by alternatively pressurizing bladders 844 and846. Once the magnetic beads are washed, the magnetic beads arerecaptured in blister 246 by activation of magnet 850, and the washsolution is then moved to blister 222. This process may be repeated asnecessary to wash the lysis buffer and sample debris from the nucleicacid-binding magnetic beads. Illustratively, three washes are done, oneeach using wash reagents in chambers 292 c, 292 d, and 292 e. However,it is understood that more or fewer washes are within the scope of thisinvention. If more washes are desired, more chambers 292 may beprovided. Alternatively, each chamber 292 may hold a larger volume offluid and activation of the plungers may force only a fraction of thevolume from the chamber upon each activation.

After washing, elution buffer stored in chamber 292 f is moved viachannel 247 to blister 248, and the magnet is retracted. The solution iscycled between blisters 246 and 248 via channel 252, breaking up thepellet of magnetic beads in blister 246 and allowing the capturednucleic acids to dissociate from the beads and come into solution. Themagnet 850 is once again activated, capturing the magnetic beads inblister 246, and the eluted nucleic acid solution is forced into blister248.

Plunger 268 h is depressed and first-stage PCR master mix from chamber292 h is mixed with the nucleic acid sample in blister 248. Optionally,the mixture is mixed by alternative activation of bladders 848 and 864,forcing the mixture between 248 and 264 via channel 253. After severalcycles of mixing, the solution is contained in blister 264, wherefirst-stage multiplex PCR is performed. If desired, prior to mixing, thesample may be retained in blister 246 while the first-stage PCR mastermix is pre-heated, illustratively by moving the first-stage PCR mastermix into blister 264 or by providing a heater adjacent blister 248. Asdiscussed above, this pre-heating may provide the benefits of hot startPCR. The instrument 800 illustrated in FIG. 8 features Peltier-basedthermal cyclers 886 and 888 which heat and cool the sample. However, itis understood that other heater/cooler devices may be used, as discussedabove. Optionally, mixing between blisters 248 and 264 may continueduring temperature cycling, with thermal cycler 886 positioned to heatand cool both blisters 248 and 264. It has been found that such mixingimproves the first-stage PCR reaction in some embodiments. Also, thermalcycling can be accomplished by varying the temperatures in two or moredifferent blisters, allowing minimal energy expenditure and maximizingthermal cycling speed. For example the temperature can be maintained at95° C. in blister 248, and 65° C. blister 264, and moving the samplebetween these blisters effectively transfers heat into and out of thesample, allowing rapid and accurate thermal cycling. Temperature cyclingis illustratively performed for 15-20 cycles, although other levels ofamplification may be desirable, depending on the application, asdiscussed above. As will be seen below, the second-stage amplificationzone 280 is configured to detect amplification in 18 second-stagereactions. Accordingly, 18 different primer-pairs may be included in thePCR reaction in blister 264.

In an alternative hot start method, pouch 210 is manufactured with theprimers provided in one of the blisters, illustratively blister 264. Inone embodiment, the primers are freeze dried separately and thenintroduced during manufacture into blister 264 as a friable pellet.Prior to first-stage PCR, illustratively the sample is eluted fromblister 246 and pushed to blister 264 to rehydrate the primer pellet.Peltier 886, which is positioned adjacent blisters 248 and 264 is heatedto 48° C., and PCR master mix is pushed to blister 248. After a hold,illustratively for 10 seconds, during which the two blisters reach 48°C., mixing between blisters 248 and 264 begins. Thus, the enzymes anddNTPs remain in blister 248 and most of the sample and the primersremain in blister 264 until the components separately have reached 48°C. It is understood, however, that the choice of 48° C. was made for usewith concurrent first-stage amplification and RT using AMV, which isactive up to 50° C. If RT is not needed or a more thermostable RT enzymeis used, then one or both of the two blisters 248 and 264 may be heatedup to 58° C., or even higher, depending on the primer meltingtemperature or other factors in a particular first-stage amplificationprotocol. It is understood that this hot start method may be used withany embodiment of the present invention.

In an alternative embodiment, to reduce the complexity of thefirst-stage PCR reaction, blister 248 may be divided into two or moreblisters. It is believed that the number of nonspecific products of amultiplex reaction goes up as the square (or possibly higher power) ofthe number of primers in the mixture, while the loss of sensitivity ofan assay is a linear function of the amount of input sample. Thus, forexample, splitting the first stage PCR into two reactions, each of halfthe volume of the single reaction of this embodiment, would reducesensitivity by two-fold but the quantity and complexity of thenonspecific reactions would be ¼ as much. If blister 248 is divided intoor more blisters, blister 264 may be divided into a number of blistersequal to the number of blisters 248. Each respective blister 248 wouldbe connected to its respective blister 264 via a respective channel 253.Each blister 264 would be provided with a pellet comprising a subset ofall primers. Sample from blister 246 would be divided across eachblister 248, each blister 248 would be sealed from all others, andthermal cycling would proceed with each pair of blisters 248 and 264, asdescribed above. After thermal cycling, the sample would be recombinedinto blister 266 or individually sent to separate sets of second-stageblisters.

After first-stage PCR has proceeded for the desired number of cycles,the sample may be diluted as discussed above with respect to theembodiment of FIG. 5A, by forcing most of the sample back into blister248, leaving only a small amount, and adding second-stage PCR master mixfrom chamber 292 i. Alternatively, a dilution buffer from 292 i may bemoved to blister 266 via channel 249 and then mixed with the amplifiedsample in blister 264 by moving the fluids back and forth betweenblisters 264 and 266. After mixing, a portion of the diluted sampleremaining in blister 264 is forced away to three-lobed blister 222, nowthe waste receptacle. If desired, dilution may be repeated severaltimes, using dilution buffer from chambers 292 j and 292 k, and thenadding second-stage PCR master mix from chamber 292 g to some or all ofthe diluted amplified sample. It is understood that the level ofdilution may be adjusted by altering the number of dilution steps or byaltering the percentage of the sample discarded prior to mixing with thedilution buffer or second-stage PCR master mix. If desired, this mixtureof the sample and second-stage PCR master mix may be pre-heated inblister 264 prior to movement to second-stage blisters 282 forsecond-stage amplification. As discussed above, such preheating mayobviate the need for a hot-start component (antibody, chemical, orotherwise) in the second-stage PCR mixture.

The illustrative second-stage PCR master mix is incomplete, lackingprimer pairs, and each of the 18 second-stage blisters 282 is pre-loadedwith a specific PCR primer pair. If desired, second-stage PCR master mixmay lack other reaction components, and these components may then bepre-loaded in the second-stage blisters 282 as well. As discussed abovewith the prior examples, each primer pair may be identical to afirst-stage PCR primer pair or may be nested within the first-stageprimer pair. Movement of the sample from blister 264 to the second-stageblisters completes the PCR reaction mixture. Control samples fromchamber 292 l are also moved to control blisters 283 via channel 267.The control samples may be positive or negative controls, as desired.Illustratively, each pouch would contain control reactions that validatethe operation of each step in the process and demonstrate that positiveresults are not the result of self-contamination with previouslyamplified nucleic acids. However, this is not practical in manyprotocols, particularly for a highly multiplexed reaction. Oneillustrative way of providing suitable controls involves spiking sampleswith a species such as baker's yeast. The nucleic acids are extractedfrom the yeast, alongside other nucleic acids. First- and second-stagePCR reactions amplify DNA and/or RNA targets from the yeast genome.Illustratively, an mRNA sequence derived from a spliced pre-mRNA can beused to generate an RNA-specific target sequence by arranging the primersequences to span an intron. A quantitative analysis of the yeast copynumber against reference standards allows substantial validation thateach component of the system is working. Negative control reactions foreach of the many second-stage assays are more problematic. It may bedesirable to run control reactions either in parallel or in a separaterun.

Activation of bladder 882 of bladder assembly 810 seals the samples intotheir respective second-stage blisters 282, 283, and activation ofbladder 880 provides gentle pressure on second-stage blisters 282, 283,to force second-stage blisters 282, 283 into contact with aheater/cooler device. A window 847 positioned over the second-stageamplification zone 280 allows fluorescence monitoring of the arrayduring PCR and during a DNA melting-curve analysis of the reactionproducts.

It is noted that the pouch 210 of FIG. 6A has several unsealed areas,such as unsealed area 255 and unsealed area 256. These unsealed areasform blisters that are not involved in any of the reactions in thisillustrative embodiment. Rather, these unsealed areas are provided inspace between the working blisters and channels. In some manufacturingprocesses, as compared to pouches that are sealed in all unused space,it has been found that fewer leaks sometimes result when unsealed areassuch as 255 and 256 are provided, presumably by reducing problematicwrinkles in the film material. Such unsealed areas optionally may beprovided on any pouch embodiment.

FIG. 8 shows an illustrative apparatus 800 that could be used with pouch210. Instrument 800 includes a support member 802 that could form a wallof a casing or be mounted within a casing. Instrument 800 also includesa second support member 804 that is optionally movable with respect tosupport member 802, to allow insertion and withdrawal of pouch 210.Movable support member 804 may be mounted on a track or may be movedrelative to support member 802 in any of a variety of ways.Illustratively, a lid 805 fits over pouch 210 once pouch 210 has beeninserted into instrument 800. In another embodiment, both supportmembers 802 and 804 may be fixed, with pouch 210 held into place byother mechanical means or by pneumatic pressure.

Illustratively, the bladder assembly 810 and pneumatic valve assembly808 are mounted on movable member 802, while the heaters 886 and 888 aremounted on support member 802. However, it is understood that thisarrangement is illustrative only and that other arrangements arepossible. As bladder assembly 810 and pneumatic valve assembly 3Q 808are mounted on movable support member 804, these pneumatic actuators maybe moved toward pouch 210, such that the pneumatic actuators are placedin contact with pouch 210. When pouch 210 is inserted into instrument800 and movable support member 804 is moved toward support member 802,the various blisters of pouch 210 are in a position adjacent to thevarious pneumatic bladders of bladder assembly 810 and the variouspneumatic pistons of pneumatic valve assembly 808, such that activationof the pneumatic actuators may force liquid from one or more of theblisters of pouch 210 or may form pinch valves with one or more channelsof pouch 210. The relationship between the blisters and channels ofpouch 210 and the pneumatic actuators of bladder assembly 810 andpneumatic valve assembly 808 are discussed in more detail below withrespect to FIGS. 9 and 10.

Each pneumatic actuator has one or more pneumatic fittings. For example,bladder 824 of bladder assembly 810 has pneumatic fitting 824 a andpneumatic piston 843 has its associated pneumatic fitting 843 a. In theillustrative embodiment, each of the pneumatic fittings of bladderassembly 810 extends through a passageway 816 in movable support member804, where a hose 878 connects each pneumatic fitting to compressed airsource 895 via valves 899. In the illustrative embodiment, thepassageways 816 not only provide access to compressed air source 895,but the passageways also aid in aligning the various components ofbladder assembly 810, so that the bladders align properly with theblisters of pouch 210.

Similarly, pneumatic valve assembly 808 is also mounted on movablesupport member 804, although it is understood that other configurationsare possible. In the illustrative embodiment, pins 858 on pneumaticvalve assembly 808 mount in mounting openings 859 on movable supportmember 804, and pneumatic pistons 843, 852, 853, and 862 extend throughpassageways 816 in movable support member 804, to contact pouch 210. Asillustrated, bladder assembly is mounted on a first side 811 of movablesupport member 804 while pneumatic valve assembly 808 is mounted on asecond side 812 of movable support member 804. However, becausepneumatic pistons 843, 852, 853, and 862 extend through passageways 816,the pneumatic pistons of pneumatic valve assembly 808 and the pneumaticbladders of bladder assembly 810 work together to provide the necessarypneumatic actuators for pouch 210.

As discussed above, each of the pneumatic actuators of bladder assembly810 and pneumatic valve assembly 808 has an associated pneumaticfitting. While only several hoses 878 are shown in FIG. 8, it isunderstood that each pneumatic fitting is connected via a hose 878 tothe compressed gas source 895. Compressed gas source 895 may be acompressor, or, alternatively, compressed gas source 895 may be acompressed gas cylinder, such as a carbon dioxide cylinder. Compressedgas cylinders are particularly useful if portability is desired. Othersources of compressed gas are within the scope of this invention.

Several other components of instrument 810 are also connected tocompressed gas source 895. Magnet 850, which is mounted on a first side813 of support member 802, is illustratively deployed and retractedusing gas from compressed gas source 895 via hose 878, although othermethods of moving magnet 850 are known in the art. Magnet 850 sits inrecess 851 in support member 802. It is understood that recess 851 canbe a passageway through support member 802, so that magnet 850 cancontact blister 246 of pouch 210. However, depending on the material ofsupport member 802, it is understood that recess 851 need not extend allthe way through support member 802, as long as when magnet 850 isdeployed, magnet 850 is close enough to provide a sufficient magneticfield at blister 246, and when magnet 850 is retracted, magnet 850 doesnot significantly affect any magnetic beads present in blister 246.While reference is made to retracting magnet 850, it is understood thatan electromagnet may be used and the electromagnet may be activated andinactivated by controlling flow of electricity through theelectromagnet. Thus, while this specification discusses withdrawing orretracting the magnet, it is understood that these terms are broadenough to incorporate other ways of withdrawing the magnetic field. Itis understood that the pneumatic connections may be pneumatic hoses orpneumatic air manifolds, thus reducing the number of hoses or valvesrequired.

The various pneumatic pistons 868 of pneumatic piston array 869, whichis mounted on support 802, are also connected to compressed gas source895 via hoses 878. While only two hoses 878 are shown connectingpneumatic pistons 868 to compressed gas source 895, it is understoodthat each of the pneumatic pistons 868 are connected to compressed gassource 895. Twelve pneumatic pistons 868 are shown. When the pouch 210is inserted into instrument 800, the twelve pneumatic pistons 868 arepositioned to activate their respective twelve plungers 268 of pouch210. When lid 805 is closed over pouch 210, a lip 806 on lid 805provides a support for fitment 29Q, so that as the pneumatic pistons 868are activated, lid 805 holds fitment 290 in place. It is understood thatother supports for fitment 290 are within the scope of this invention.

A pair of heating/cooling devices, illustratively Peltier heaters, aremounted on a second side 814 of support 802. First-stage heater 886 ispositioned to heat and cool the contents of blister 264 for first-stagePCR. Second-stage heater 888 is positioned to heat and cool the contentsof second-stage blisters 282 and 283 of pouch 210, for second-stage PCR.It is understood, however, that these heaters could also be used forother heating purposes, and that other heaters may be included, asappropriate for the particular application.

If desired, a feedback mechanism (not shown) may be included ininstrument 800 for providing feedback regarding whether the sample hasactually been forced into a particular blister. Illustrative feedbackmechanisms include temperature or pressure sensors or optical detectors,particularly if a fluorescent or colored dye is included. Such feedbackmechanisms illustratively may be mounted on either of support members802 or 804. For example, a pressure sensor may be mounted on support 802adjacent the location of blister 264. When the sample is supposedlymoved to blister 264, if the pressure sensor is depressed, then sampleprocessing is allowed to continue. However, if the pressure sensor isnot depressed, then sample processing may be stopped, or an errormessage may be displayed on screen 892. Any combination or all of theblisters may have feedback mechanisms to provide feedback regardingproper movement of the sample through the pouch.

When fluorescent detection is desired, an optical array 890 may beprovided. As shown in FIG. 8, optical array 890 includes a light source898, illustratively a filtered LED light source, filtered white light,or laser illumination, and a camera 896. A window 847 through movablesupport 804 provides optical array 890 with access to second-stageamplification zone 280 of pouch 210. Camera 896 illustratively has aplurality of photodetectors each corresponding to a second-stage blister282, 823 in pouch 210. Alternatively, camera 896 may take images thatcontain all of the second-stage blisters 282, 283, and the image may bedivided into separate fields corresponding to each of the second-stageblisters 282, 283. Depending on the configuration, optical array 890 maybe stationary, or optical array 890 may be placed on movers attached toone or more motors and moved to obtain signals from each individualsecond-stage blister 282, 283. It is understood that other arrangementsare possible.

As shown, a computer 894 controls valves 899 of compressed air source895, and thus controls all of the pneumatics of instrument 800. Computer894 also controls heaters 886 and 888, and optical array 890. Each ofthese components is connected electrically, illustratively via cables891, although other physical or wireless connections are within thescope of this invention. It is understood that computer 894 may behoused within instrument 890 or may be external to instrument 890.Further, computer 894 may include built-in circuit boards that controlsome or all of the components, and may also include an externalcomputer, such as a desktop or laptop PC, to receive and display datafrom the optical array. An interface 893, illustratively a keyboardinterface, may be provided including keys for inputting information andvariables such as temperatures, cycle times, etc. Illustratively, adisplay 892 is also provided. Display 892 may be an LED, LCD, or othersuch display, for example.

FIG. 9 shows the relationship between bladder assembly 810 and pouch 210during operation of instrument 800. Bladder assembly comprisessub-assemblies 815, 817, 818, 819, and 822. Because bladder 809 ofbladder sub-assembly 815 is large, bladder sub-assembly 815illustratively has two pneumatic fittings 815 a and 815 b. Bladder 809is used to close off chambers 292 (as shown in FIG. 6A) from the plasticfilm portion 217 of pouch 210. When one of the plungers 268 isdepressed, one or both of pneumatic fittings 815 a and 815 b permitbladder 809 to deflate. After the fluid from one of the chambers 292passes through, bladder 809 is re-pressurized, sealing off channels 214,236, 245, 247, and 249. While illustrative bladder sub-assembly 815 hasonly one bladder 809, it is understood that other configurations arepossible, illustratively where each of channels 214, 236, 245, 247, and249 has its own associated bladder or pneumatic piston. Bladdersub-assembly 822 illustratively comprises three bladders 824, 826, and828. As discussed above, bladders 824, 824, and 828 drive thethree-lobed blister 222 for cell lysis. As illustrated, bladders 824,826, and 828 are slightly larger than their corresponding blisters 224,226, 228. It has been found that, upon inflation, the surface of thebladders can become somewhat dome-shaped, and using slightly oversizedbladders allows for good contact over the entire surface of thecorresponding blister, enabling more uniform pressure and betterevacuation of the blister. However, in some circumstances, completeevacuation of individual blisters may or may not be desired, and largeror smaller-sized bladders may be used to control the blister volumeevacuated. Bladder sub-assembly 817 has four bladders. Bladder 836functions as a pinch-valve for channel 236, while bladders 844, 848, and866 are configured to provide pressure on blisters 244, 248, and 266,respectively. Bladder sub-assembly 818 has two bladders 846 and 864,which are configured to provide pressure on blisters 246 and 264,respectively. Finally, bladder sub-assembly 819 controls second-stageamplification zone 280. Bladder 865 acts as a pinch valve for channels265 and 267, while bladder 882 provides gentle pressure to second-stageblisters 282 and 283, to force second-stage blisters into close contactwith heater 888. While bladder assembly 810 is provided with fivesub-assemblies, it is understood that this configuration is illustrativeonly and that any number of sub-assemblies could be used or that bladderassembly 810 could be provided as a single integral assembly.

FIG. 10 similarly shows the relationship between pneumatic valveassembly 808 and pouch 210 during operation of instrument 800. Ratherthan bladders, pneumatic valve assembly 808 has four pneumatic pistons842, 852, 853, and 862. These pneumatic pistons 842, 852, 853, and 862,each driven by compressed air, provide directed pressure on channels242, 252, 253, and 262. Because the pistons are fairly narrow indiameter, they can fit between bladder sub-assembly 817 and bladdersub-assembly 818 to provide pinch valves for channels 242, 252, 253, and262, allowing channels 242, 252, 253, and 262 to be fairly short.However, if desired, pneumatic pistons 842, 852, 853, and 862 could bereplaced by bladders, which may be included in bladder assembly 810,obviating the need for pneumatic valve assembly 808. It is understoodthat any combination of bladders and pneumatic pistons are within thescope of this invention. It is also understood that other methods ofproviding pressure on the channels and blisters of pouch 210, as areknown in the art, are within the scope of this invention.

FIG. 12A shows a pouch 510 that is similar to pouch 210 of FIG. 6A.Fitment 590, with entry channels 515 a through 5151, is similar tofitment 290, with entry channels 215 a through 215 l. Blisters 544, 546,548, 564, and 566, with their respective channels 538, 543, 552, 553,562, and 565 are similar to blisters 244, 246, 248, 264, and 266, withtheir respective channels 238, 243, 252, 253, 262, and 265 of pouch 210.The channels 245, 247, and 249 of pouch 210 have been somewhatreconfigured as channels 545 a-c, 547 a-b, and 548 a-c on pouch 510; therespective channels of 510 are somewhat shorter than their counterpartchannels on pouch 210. However, it is understood that channelconfigurations are illustrative only, and that various channelconfigurations are within the scope of this invention.

There are two main differences between pouch 510 of FIG. 12A and pouch210 of FIG. 6A. First, three-lobed blister 222 has been replaced bylysis blister 522. Lysis blister 522 is configured for vortexing viaimpaction using rotating blades or paddles 21 attached to electric motor19, as shown in FIG. 2C. Since this method of lysing does not rely onalternating pressure of pneumatic pistons, only a single-lobed blisteris shown. Because lysis blister 522 has only a single lobe, bothchannels 514 and 536 lead to the single lobe of lysis blister 522. It isunderstood that lysis blister 522 may be used in any of the pouchembodiments described herein. It is further understood that lysisassembly 810 illustratively may be modified to replace bladders 824,826, and 828 of bladder sub-assembly 822 with a single bladderconfigured for blister 522. Conversely, a three-lobed blister, asdescribed in various other embodiments above, may be used in pouch 510.Lysis blister 522 may be provided with an optional reinforcing patch523, illustratively attached using adhesive or lamination to theexterior surface of lysis blister 522. Reinforcing patch 523 aids inminimizing tearing of pouch 510 due to repeated contact by paddles 21.FIG. 12B shows an electric motor, illustratively a Mabuchi RC-280SA-2865DC Motor (Chiba, Japan), mounted on second support member 804. In oneillustrative embodiment, the motor is turned at 5,000 to 25,000 rpm,more illustratively 10,000 to 20,000 rpm, and still more illustrativelyapproximately 15,000 to 18,000 rpm. For the Mabuchi motor, it has beenfound that 7.2V provides sufficient rpm for lysis. It is understood,however, that the actual speed may be somewhat slower when the blades 21are impacting pouch 510. Other voltages and speeds may be used for lysisdepending on the motor and paddles used. Optionally, controlled smallvolumes of air may be provided into the bladder adjacent lysis blister522. It has been found that in some embodiments, partially filling theadjacent bladder with one or more small volumes of air aids inpositioning and supporting lysis blister during the lysis process.Alternatively, other structure, illustratively a rigid or compliantgasket or other retaining structure around lysis blister 522, can beused to restrain pouch 510 during lysis.

The second main difference between pouch 510 of FIG. 12A and pouch 210of FIG. 6A is that the blisters 281, 282, and 283 of second-stageamplification zone 280 have been replaced by high density array 581 insecond-stage amplification zone 580. High density array 581 comprises aplurality of second-stage wells 582, illustratively 50 or more wells,and even more illustratively 120 or more wells. Embodiments with moresecond-stage wells 582, illustratively about 200, about 400, or evenabout 500 or more are within the scope of this invention. Otherconfigurations are within the scope of this invention as well.Additional second-stage wells 582 may be added by making wells 582smaller, by making high density array 581 larger, or a combinationthereof. For second-stage PCR, each of the wells may contain a pair ofprimers. It is understood that one or more wells may be used forpositive or negative controls.

Cross-contamination between wells as the wells are filled with thediluted first-stage amplification product in blister 566 can be a majorproblem. Cross-contamination was controlled in pouch 210 by filling eachsecond-stage blister through a separate branch of channel 265 and thensealing with bladder 882, illustrated in FIG. 9. With high density array581, wherein fluid may fill some or all of blister 584,cross-contamination between wells must also be controlled. In oneembodiment, the second-stage PCR primers may be bound covalently ornon-covalently to the inside surface of each well, thus functioning muchlike a PCR chip. However, in many embodiments it is desirable to controlcross-contamination between wells without tethering the PCR primers tothe wells. Controlling cross-contamination between wells can bedifficult in an embodiment wherein the fluid from blister 566 is movedto wells 582 by flowing across a first surface 581 a of high densityarray 581.

There are several desirable features for successful loading of thesecond-stage amplification zone 580. First, it is desirable that theincoming fluid from blister 566 fill substantially all of the wells 582to substantially the same level. An unfilled well would produce a falsenegative signal. Second, it is desirable that the process of filling thewells 582 should not cause the primers in the well to leak out. Loss ofprimers from one well can limit the efficiency of the PCR reaction inthat well and can contaminate neighboring wells. Third, after the wells582 have been filled and PCR started, it is desirable that the wells becompletely sealed from each other. Amplicon leakage out of one well andinto another well can lower signal in the first well and raise signal inthe second well, potentially leading to a false negative in the firstwell and a false positive in the second well. Further, for certain kindsof controls, it is important that amplicon generated in one well notenter another well where it can be further amplified.

Solutions to this problem include use of a barrier layer. In oneexample, the barrier layer is a physical barrier that is provided toallow for rapid loading of the wells and for rapid sealing from the bulkfluid. In another example, combined chemical and physical barriers areused, wherein the physical barrier is used to seal the wells and thenthe chemical barrier conditionally releases the oligonucleotide primersinto the well solution, for example by heating, slow release, orenzymatic digestion. Well depth or channel length to each well also maybe used to control release of the reagents from the wells. Other meansfor loading high density array 581 are possible.

FIGS. 12-14 show an illustrative embodiment of second-stage 580 using aphysical barrier. Sandwiched between first layer 518 and second layer519 of pouch 510 is high density array 581, with wells 582. As best seenin FIG. 13, pierced layer 585, with piercings 586, is provided on oneside of high density array 581 to act as the physical barrier, and asecond layer 587, is provided on the opposite side of high density array581 to form the bottom of wells 582. Illustratively, pierced layer 585and second layer 587 are plastic films that have been sealed to highdensity array 581, illustratively by heat sealing, although it isunderstood that other methods of sealing may be employed. It is alsounderstood that the material used for high density array 581 and thematerial used for pierced layer 585 and second layer 587 should becompatible with each other, with the sealing method, and with thechemistry being used. When used for PCR, examples of compatible plasticsthat can used for high density array 581 and can be heat-sealed are PE,PP, Monprene®, and other block copolymer elastomers. If fluorescent dyesare used in the detection chemistry, it may be desirable for highdensity array 581 to be formed from black or other relativelyfluorescently opaque materials, to minimize signal bleed from one well582 to its neighboring wells and for at least one of layers 585 and 587to be relatively fluorescently transparent. For pierced layer 585 andsecond layer 587, laminates of a strong engineering plastic such asMylar® or PET with heat-sealable plastic layers such as PE, PP andDupont Surlyn® may be used. For adhesive-based systems, rigidengineering plastics such as PET or polycarbonate may be used to formhigh density array 581 and films of PCR-compatible plastics are thenused as pierced layer 585 and second layer 587. In one illustrativeembodiment, high density array 581 is formed of black PE, a compositepolyethylene/PET laminate (or Xerox® PN 104702 hot laminating pouchmaterial) is used for pierced layer 585 and second layer 587 which areheat sealed to high density array 581, and composite polypropylene/PETis used for first and second layers 518, 519 of pouch 510.

It is understood that piercings 586 align with wells 582. It is alsounderstood that piercings 586 are small enough that, absent some force,fluid does not readily flow through piercings 586. Illustrativepiercings may be 0.001-0.1 mm, more illustratively 0.005-0.02 mm, andmore illustratively about 0.01 mm. In the illustrative embodiment,second-stage amplification zone 580 is provided under vacuum, such thatwhen fluid is received from blister 566, the vacuum draws fluid throughpiercings 586 into each well 582. Once the wells 582 are filled, a forceis no longer present to force fluid into or out of the wells 582. Abladder adjacent second-stage amplification zone 580 (not shown, butsimilar in position to bladders 880/882) may then be activated to pressfirst layer 518 against high density array 581 and seal fluid into thewells 582. While first layer 518 of pouch 510 is used to seal the wells582, it is understood that an optional sealing layer may be providedbetween pierced layer 585 and first layer 510.

In one illustrative example, second-stage amplification zone 580 may beprepared as follows. High density array 581 may be prepared by firstpunching, molding, or otherwise forming an array of wells 582 in aplastic sheet (illustratively 0.1 to 1 mm thick). The wells may form anyregular or irregular array that is desired, and may have a volumeillustratively of 0.05 μl to 20 μl, and more illustratively of 0.1 μl to4 μl. One of layers 585 or 587 is then laminated to a first surface 581a of high density array 581, illustratively by heat or adhesive. Asshown in FIG. 14, pierced layer 585 is applied to first surface 581 a.Reagents 589, illustratively elements of the chemistry of the array thatare unique, such as PCR primer pairs, are then spotted into the wellseither manually by pipetting, or automatically (illustratively using x/ypositionable spotters such as pin-spotters, dot-matrix printers,small-volume automatic pipettes, or micro-fluidic micro-contactspotters). After the reagents 589 have been dried in each well 582, thesecond of layers 585 or 587 is applied to the second surface 581 b ofarray 581. Layer 585 is pierced using an array of small diameter needlesto form piercings 586. Piercings 586 may be formed either before orafter layer 585 has been fixed to array 581. It is understood thatspotting can be done on either layer 585 before or after piercing or onlayer 587. Spotting an array with holes pre-pierced has not shown toleak substantially and offers the advantage that the needles used forpiercing are not contaminated by touching the spotted reagents.Alternatively, to minimize the possibility of leakage, and to positionthe spotted reagents at the most distant location in the array, it maybe desirable to spot the reagents 589 onto second layer 587, seal thearray 581 with layer 585, and then pierce layer 585. In an illustrativeexample, reagents are spotted onto second layer 587 using a GeSiMA060-324 Nano-Plotter 2.1/E (Grosserkmannsdorf, Germany). Using such aspotter, multiple arrays may be spotted simultaneously.

Once spotted and pierced, array 581 is placed inside layers 518 and 519of pouch 510 and sealed in place, illustratively by either heat sealing,using an adhesive, ultrasonically welding, mechanical closure, or othermeans of enclosing array 581 inside pouch 51Q within blister 584. It isunderstood that blister 584 is fluidly connected to blister 566 viachannel 565, and that liquid can flow from channel 565 into blister 584and over piercings 586. In one illustrative example, when blister 584 isformed, care is taken to allow a path for air to escape. This can beaccomplished by “waffling” the inside surface of first layer 518adjacent to second-stage amplification zone 580 to imprint the filmmaterial with a pattern of slightly raised texture. This allows air andliquid to pass along the surface of pierced layer 585, and better allowsliquid to reach and fill all of wells 582. The pouch 510 is then placedinside a vacuum chamber and evacuated. Illustratively, when the pressurehas reached approximately 0.3 millibars, a pneumatic cylinder inside thevacuum chamber is actuated, driving down a plunger into fitment 590 toseal channel 567, thereby cutting the path from the array inside thesealed pouch, and the vacuum chamber. A plurality of other plungers arealso driven into fitment 590 to seal the various entry channels 515. Thepouch is removed from the vacuum chamber and may be packaged forlong-term storage in a vacuum-bag.

Pouch 510 may be used in a manner similar to pouch 210. Because array581 is packaged in vacuum, when liquid is moved from blister 566 tosecond-stage amplification zone 580, the liquid sample is drawn throughpiercings 586 and into wells 582. Excess liquid is forced away byinflating a pneumatic bladder over the array and thermal cycling isaccomplished as described above, illustratively by heating and cooling aPeltier element pressed against one side of the array.

As mentioned above, pierced layer 585 may be replaced by a variety ofsuitable physical or chemical barriers. In one illustrative embodimentusing a chemical barrier, pierced layer 585 is omitted, and reagents 589are spotted into wells 582 in a buffer that dissolves relatively slowly.Illustratively, reagents 589 that contain polymers such as PEG, Ficollor polysorbate 20 or sugars such as sucrose, trehalose or mannitol inappropriate concentrations will be compatible with the second-stage PCRreaction and may dissolve more slowly than primers spotted solely inwater or Tris/EDTA. The primers spotted in one of these buffers may beair dried into the wells 582, as described above (it is understood thatin such an embodiment, second layer 587 is affixed to high density array581 for spotting). These same polymers may be used in lyophilization ofenzyme reagents (e.g. the enzymes and buffers used in PCR) to form anopen matrix containing the stabilized enzymes. Thus, the primers spottedin these buffers can be lyophilized in place in the wells 582, leadingto slower but potentially more complete rehydration than with airdrying. When pouch 510 is used, the fluid from blister 566 is driveninto the well by vacuum or pressure and starts to dissolve the primermix. By selecting a buffer that dissolves suitably slowly, when thebladder adjacent second-stage amplification zone 580 is actuated, thecontents of each well 582 is sealed therein prior to any substantialcross-contamination.

Another embodiment uses a matrix that does not dissolve untilsecond-stage amplification zone 580 is heated above a predeterminedtemperature. One example of such a matrix is low melt agarose such asGenePure LowMelt Agarose (ISC Bioexpress). In one example, a 1.5%solution of this agarose melts at 65° C. and gels at 24-28° C. Prior tospotting, reagents 589 illustratively may be warmed to 50° C. and mixedwith this agarose that had already been melted and then spotted intowells 582 in a small volume (illustratively 100 to 500 nl). To keep themixture liquid during spotting, this may have to be done in a cabinetheated above the melting temperature of the agarose. Alternatively, itmay be possible to pipette dilute solutions of the agarose withoutmelting. After the agarose/reagent mixture is spotted, the high densityarray 581 is dried. This can be a simple air drying or theprimer-agarose mixture can contain the sugars and polymers listed aboveso that the reagents can be freeze dried. When pouch 510 is used forPCR, second-stage amplification zone 580 may be heated, illustrativelyto 55° C., as the fluid from blister 566 is moved into high densityarray 581. At this temperature, the agarose does not melt so the primersare not released into solution. Once high density array 581 is filled,the corresponding bladder is inflated to seal the wells. When thetemperature rises above 65° C. in the first denaturation step of thefirst PCR cycle, the agarose containing the primers melts, releasing theprimers into the master mix. Illustratively, thermal cycling never goesbelow 60° C. (or other melting temperature for the agarose) so that theagarose does not gel during thermal cycling. Furthermore, in theillustrative instrument 800 of FIG. 8, the repeated temperature cyclingis driven by heater 888, which is located on one side of the pouch. Itis expected that there will often be a temperature gradient across thePCR solution in wells 582, which should facilitate mixing of the primersby convective fluid flow. Wax may also be used in a similar embodiment.

In a further embodiment, the primers may be conditionally bound to thewells 581, with subsequent releasing of the primers into solution afterthe wells 581 have been filled. Depending upon how the primers areattached to the plastic substrate, the primers may be cleaved using heat(illustratively during the first cycle of the PCR reaction), light(illustratively irradiating through window 847), chemicals (e.g.dithiothreitol together with heat will reduce disulfide bonds that maybe used to link primers to the wells), or enzymes (e.g. site specificproteases such at Tissue Plasminogen Activator can be used to cleave theproper peptide linker attaching primers to the substrate).

In yet another embodiment, a DNase may be injected into second-stageamplification zone 580 subsequent to amplification, to minimize furtherany potential risk of contamination.

It is understood that second-stage amplification zone 580 has beendescribed herein for use with PCR. However, other uses for pouch 510 andsecond-stage amplification zone 580 are within the scope of thisinvention. Further, it is understood that second-stage amplificationzone 580 may be used with or without nucleic acid extraction and a firststage PCR amplification zone. Finally, it is understood thatsecond-stage amplification zone 580 may be used with any of the pouchembodiments described herein.

EXAMPLE 1: NESTED MULTIPLEX PCR

A set of reactions was run in a pouch 110 of FIG. 5A, on an instrumentsimilar to instrument 800 but configured for pouch 110. To show celllysis and effectiveness of the two-stage nucleic acid amplification, 50μL each of a live culture of S. cerevisaie and S. pombe at log phase wasmixed with 100 μL of a nasopharyngeal aspirate sample from a healthydonor to form the sample, then mixed with 200 μL lysis buffer (6Mguanidine-HCl, 15% TritonX 100, 3M sodium acetate. 300 μL of the 400 μLsample in lysis buffer was then injected into chamber 192 a of pouch110.

The pouch 110 was manufactured with 0.25 g ZS beads sealed inthree-lobed blister 122. Second-stage primers, as discussed below, werealso spotted in blisters 181 and 182 during manufacture of pouch 110.The pouch 110 was loaded as follows:

115 a sample and lysis buffer, as described above,

115 b magnetic beads in the lysis buffer,

115 d-e wash buffer (10 mM sodium citrate),

115 g elution buffer (10 mM Tris, 0.1 mM EDTA)

115 h first-stage PCR buffer:

-   -   0.2 mM dNTPs    -   0.3 μM each primer:        -   Sc1: primers configured for amplifying a portion of the YRA1            nuclear protein that binds to RNA and to MEX67p of S.            cerevisaie. The primers are configured to amplify across an            intron such that amplification of cDNA (mRNA            reverse-transcribed via M-MLV) yields a 180 bp amplicon.        -   Sc2: primers configured for amplifying a 121 bp region of            the cDNA of MRK1 glycogen synthase kinase 3 (GSK-3) homolog            of S. cerevisaie.        -   Sc3: primers configured for amplifying a 213 bp region of            the cDNA of RUB1 ubiquitin-like protein of S. cerevisaie.        -   Sp1: primers configured for amplifying a 200 bp region of            the cDNA of suc1-cyclin-dependent protein kinase regulatory            subunit of S. pombe.        -   Sp2: primers configured for amplifying a 180 bp region of            the cDNA of sec14-cytosolic factor family of S. pombe.    -   PCR buffer with 3 mM MgCl₂ (without BSA)    -   50 units M-MLV    -   4.5 units Taq:Antibody    -   100 units RNAseQut

115 j-k second-stage PCR buffer

-   -   0.2 mM dNTPs    -   1×LC Green® Plus (Idaho Technology)    -   PCR buffer with 2 mM MgCl₂ (with BSA),    -   4.5 units Taq

115 l second-stage PCR buffer with a sample of the first-stageamplicons.

During manufacture, second-stage blisters 181 and 182 were spotted withnested second-stage primers. Each blister was spotted with one primerpair in an amount to result in a final concentration of about 0.3 μMonce rehydrated with the second-stage PCR buffer. The second-stagenested primers are as follows:

-   -   Sc1: primers configured for amplifying an 80 bp fragment of the        Sc1 cDNA first-stage amplicon.    -   Sc2: primers configured for amplifying a 121 bp fragment of the        Sc1 cDNA first-stage amplicon.    -   Sc3: primers configured for amplifying a 93 bp portion of the        Sc1 cDNA first-stage amplicon.    -   Sp1: primers configured for amplifying a 99 bp portion of the        Sc1 cDNA first-stage amplicon.    -   Sp2: primers configured for amplifying a 96 bp portion of the        Sc1 cDNA first-stage amplicon.        There is no overlap between the first-stage and second stage        primer pairs for any of the targets. Each pair of primers was        spotted into one negative control blister 181 and two        second-stage blisters 182, so that each second-stage        amplification would be run in duplicate, each duplicate with a        negative control.

After loading, activation of the plunger associated with entry channel115 a moved the sample to three-lobed blister 122, activation of theplunger associated with entry channel 115 b moved the magnetic beads toreservoir 101, activation of the plungers associated with entry channels115 d-e moved wash buffer to reservoirs 102 and 103, activation of theplunger associated with entry channel 115 g moved elution buffer toreservoir 104, activation of the plunger associated with entry channel115 h moved first-stage PCR buffer to reservoir 105, activation of theplungers associated with entry channels 115 j-k moved second stage PCRbuffer to reservoirs 106 and 107, and activation of the plungerassociated with entry channel 115 l moved the positive control(second-stage PCR buffer with a sample of previously preparedfirst-stage amplicon) to reservoir 108. In this present example, theplungers associated with entry channels 115 a and 115 b were depressedprior to loading the pouch 110 into the instrument. All other plungerswere depressed sequentially in the instrument during the run, and fluidswere moved to reservoirs 102 through 108 as needed.

Once pouch 110 was placed into the instrument, and beating took placefor ten minutes in the presence of ZS beads, as described above. Oncecell lysis was complete, reservoir 101 was compressed and nucleic acidbinding magnetic beads from reservoir 101 were forced into three-lobedblister 122, where the beads were mixed gently and allowed to incubatefor 5 minutes.

The sample-bead mixture was then moved to blister 144, where themagnetic beads were captured via activation of the magnet. Once themagnet was deployed, bladders adjacent blister 144 were pressurized toforce fluids back to three-lobed blister 122. The captured beads werethen washed as described above, using the wash solution from reservoirs102 and 103. Following washing, the beads were once again captured inblister 144 via activation of the magnet, and the elution buffer storedin reservoir 104 is moved to blister 144, where, after a 2 minuteincubation, the nucleic acids eluted from the beads are then moved toblister 161, as discussed above.

In blister 161, the nucleic acid sample is mixed with first-stage PCRmaster mix from reservoir 105. The sample is then held at 40° C. for 10minutes (during which time M-MLV converts mRNA to cDNA), then 94° C. for2 minutes (to inactivate the M-MLV and remove antibody from taq).Thermal cycling is then 20 cycles of 94° C. for 10 second and 65° C. for20 seconds.

Subsequent to first-stage amplification, the sample is dilutedapproximately 100-fold using the second-stage PCR master mix fromreservoir 106. The sample is then moved to blisters 182, which werepreviously spotted with the second-stage primers, as discussed above.Second-stage PCR buffer was moved from reservoir 181 to negative controlblisters 181, and the positive control mixture was moved to blisters 183from reservoir 108. The samples were denatured for 30 seconds at 94° C.,then amplified for 45 cycles of 94° C. for 5 seconds and 69° C. for 20seconds.

As can be seen in FIG. 11, all target amplicons and the positive controlshowed amplification, while none of the negative controls showedamplification. Each sample was run in replicates. The replicates eachshowed similar amplification (data not shown).

It is understood that the S. cerevisaie and S. pombe targets areillustrative only and that other targets are within the scope of thisinvention.

EXAMPLE 2: HIGH DENSITY PCR

The above example uses pouch 110 of FIG. 5A. Pouch 110 has five negativecontrol blisters 181, five positive control blisters 183, and ten lowvolume sample blisters 182. Pouch 210 of FIG. 6A increased the number oflow volume sample blisters 282 to 18. However, high density array 581 ofpouch 510, shown in FIG. 12A can have 120 or more second-stage wells582. This increase in the number of second-stage reactions enables awide set of potential diagnostic and human identification applicationswithout the need to increase the size of the pouch and its instrument.Various examples are described herein.

In one example, it is known that standard commercial immunofluorescenceassays for the common respiratory viruses can detect seven viruses:Adenovirus, PIV1, PIV2, PIV3, RSV, Influenza A, and Influenza B. A morecomplete panel illustratively would include assays for additional fiveviruses: coronavirus, human metapneumovirus, BOCAvirus, Rhinovirus andnon-HRV Enterovirus. For highly variable viruses such as Adenovirus orHRV, it is desirable to use multiple primers to target all of thebranches of the virus' lineage (illustratively 4 outer and 4 innerprimer sets respectively). For other viruses such as coronavirus, thereare 4 distinct lineages (229E, NL63, OC43, HKU1) that do not vary fromone season to another, but they have diverged sufficiently enough thatseparate primer sets are required. The illustrative complete respiratoryvirus panel would also target the SARS coronavirus, possibly the avianinfluenza HA and N subtypes, and possibly others. Finally, some of therespiratory viruses show such a high rate of sequence variation that itwould be beneficial to create more than one nested PCR assay for eachsuch virus, thereby minimizing the chance of false negative results dueto sequence variation under the primers. When all of the primer setsdescribed herein are included, such a respiratory virus panel could have80 or more specific amplicons in the second-stage amplification. Thehigh density array 581 could easily accommodate such a panel in a singlepouch 510.

A second application of the high density array 581 of pouch 510 would beto determine the identity and the antibiotic resistance spectrum of themulti-drug resistant bacteria isolated from infected patients. Currentmethods require several days to culture the organism and empiricallytest individual drug resistance profiles. During the time it takes toreceive the results, physicians will often administer broad-spectrumantibiotics, which leads to an increase in multi-drug resistantbacteria. PCR primers have been developed to detect the geneticdeterminants of antibiotic resistance (the antibiotic resistance genesthemselves). However because of the large number of variants of some ofthese genes, a large number of amplicons is required for a completedetermination of the resistance profile. Hujer et. al. describe a panelof 62 PCR assays to identify the resistance genes present inAcinetobacter isolates. Again, the high density array 581 could easilyaccommodate such a panel in a single pouch.

A third example of the utility of the high density array is in the fieldof human identification, illustratively for forensic identification ofhuman remains and for paternity testing. Most of the market in humanidentification is dominated by systems that analyze short tandem repeatsequences (STRs). This analysis has generally required separating therepeats by size, using e.g. capillary electrophoresis. The specializedlaboratory equipment used for this purpose has generally not been fieldportable. There is growing interest in using Single NucleotidePolymorphisms (SNPs) for identity testing, as there are a large set oftechniques for identifying SNPs and some of these are amenable to fielduse. Sanchez et al. have published a set of 52 well-characterized SNPsthat collectively give a very low probability of matching twoindividuals by chance (a mean match probability of at least 5.0×10⁻¹⁹).In practice, it may take two amplicons for each SNP to accurately typeeach locus (see, e.g., Zhou et al.). Thus one pouch 510 with 104second-stage wells 582 could completely type an individual at all of the52 SNP loci.

It is understood that there are cost and workflow advantages gained bycombining assays from different diagnostic applications into one pouch.For example the complete respiratory virus panel could be combined withthe bacterial identification panel. These combinations could simplifymanufacturing, since there are fewer types of pouches to assemble. Theycould also simplify the work of the end user, as there are fewerspecific types of pouches that need to be stocked in a clinic, and alsoreducing the chance of using the wrong pouch for a particular clinicalsample. For some applications, these advantages could offset an increasecost of manufacturing the pouch having a greater number of primer pairs.Thus one pouch 510 with 100 or more second-stage wells 582 could be usedto accommodate multiple panels of assays.

EXAMPLE 3: PROCESS CONTROLS

Controls for highly multiplexed assays can be problematic, especially inclinical diagnostic settings where quality must compete with cost pertest. The high-density array 582 of pouch 510 potentially increases thisproblem because of the increased number of diagnostic targets that canbe assayed in a single run. Various types of controls are discussedherein.

Illustrative process controls include mixing an intact organism, forexample an organism containing an RNA target, into the patient samplebefore injecting the sample into the pouch. Such a target could be anintact RNA bacteriophage (MS2 or Qβ) or an intact plant RNA virus(Tobacco Mosaic Virus) or an mRNA present in an intact yeast. Outerprimers specific for the RNA target would be present in the first-stagePCR and a well 582 containing the inner primers would be present in thehigh density array. Detection of amplification product in this well 582confirms that all of the steps of the process are working correctly. Apost-second-stage amplification melt curve could also be used to verifythat the correct specific product was made. The crossing point (“Cp”)determined from an amplification curve could be used to give aquantitative measure of the integrity of the reagents. For example theCp can be compared to that of other pouches from the same lot run at adifferent time. While an intact organism is used, it is understood thatpurified or isolated nucleic acids may be used if it is not important totest for lysis. In other situations, it may be desirable to use thecontrol to test only the later steps of the analysis. For example,spiking a natural or synthetic nucleic acid template into a well in thehigh density array along with the cognate primers could be used to testthe second-stage PCR reaction, and spiking a nucleic acid template intothe first-stage PCR with the appropriate primers in the first-stage PCRamplification mixture and in a well 582 of the second-stageamplification zone will test both the first- and second-stage PCRreactions.

Process controls such as described above do not test the integrity ofthe primers specific to the target amplicons. One example of a positivecontrol that tests the integrity of the specific primers uses a mixtureof nucleic acids, illustratively synthetic RNAs, as stability andvariability often can be better controlled and these sequences cannot bepresent due to environmental contamination, wherein the mixture containsa nucleic acid for each of the primers present in the particular pouch.In a diagnostic setting, this positive control could be used at the endof a run of pouches used to test patient samples. The mixture isinjected into a pouch, illustratively from the same lot as those usedfor the patient samples, and success is defined by all of the targetamplicons providing a positive result. Negative controls can be done inthe same way; at the end of a run of pouches used to test patientsamples, water or buffer could be injected into a pouch and successdefined by all of the target amplicons providing a negative result.

Individual workflow and protocols in a diagnostic lab may be used todetermine the number of patient sample pouches run before the controlpouches described above are run. Regardless of how frequently orinfrequently the control pouches are run, these controls add to the timeand cost of the total system. For this reason, it would be useful tomake the controls internal to the pouch. The structure of the highdensity array 581 allows for the following novel approach to negativecontrols. In this example, a nucleic acid, illustratively a syntheticamplicon, is spiked into one of the wells 582 a of the high densityarray 581. Primers to amplify this sequence are spiked into this well582 a and into two other wells 582 b and 582 c spaced across the array.Illustratively, the amplicon sequence and primers are artificial anddesigned so that none of the primers used will amplify another target bychance.

When a clean, uncontaminated pouch 510 is run in instrument 800, thewell 582 a containing the synthetic target will generate amplicon andtherefore be called positive. The two other wells 582 a, 582 b thatcontain the corresponding primers should not amplify anything in thesample and thus be called negative. Pouch 510 may be treated further,for additional controls. Illustratively, bladder 880/882 holding thehigh density array against heater 888 is then depressurized and thecontents of the wells 582 are mixed. In one illustrative method, thecontents of the wells 582 are mixed as follows: heater 888 is used tocycle the temperature of the high density array above and below theboiling point of the buffer for a short time (for example three cyclesof 85° C. for 10 sec then 105° C. for to 20 sec). Bubbles of steamgenerated in the wells 582 of high density array 581 should force thecontents of wells 582 out into the second-stage amplification zoneblister 580. Optionally, the contents of the second-stage amplificationzone 580 may be mixed with the contents of rest of the pouch 510 byusing the bladders to move liquid from one end of the pouch 510 to theother. The purpose of these steps is to mix the specific contaminationcontrol amplicon, along with any specific target amplicons throughoutthe pouch.

If the user accidentally opens a pouch after it has been run in thisfashion, then both specific target amplicons and the contaminationcontrol amplicon will be released. If trace amounts of these nucleicacids contaminate a later pouch run, the instrument may detect thecontamination event, as the wells 582 b, 582 c that contained only theprimers specific for the synthetic amplicon will score positive.Software in the instrument will alert the user and the results of therun will be flagged as suspect.

In another method to control contamination, at the end of a run, a DNAdegrading chemical or enzyme may be added to destroy substantially allof the DNA products of the first- and second-stage PCR reactions.Illustratively, this can be done in a way similar to the contaminationdetection method described above, by heating the contents of thesecond-stage array to above the local boiling temperature, thus drawingthe amplified sample out of the wells 582 of the array 851, mixing theheated liquid with the diluted contents of the 1^(st) stage reaction,adding an aliquot of a DNA degrading substance, illustratively throughentry channel 515 k, either with or without cooling the mixture, andallowing the DNA degrading reaction to incubate until substantially allof the DNA produced in the PCR reaction has been destroyed. This can beaccomplished using DNAses, acids, or oxidants, as are known in the art.

It is understood that any of the contamination controls described hereinmay be used independently or in any combination thereof.

EXAMPLE 4: BACTERIAL IDENTIFICATION PANEL

The above instruments and pouches allow for a large number of reactionsin a single, sealed environment. This is particularly true of pouch 510,which may optionally have 100 or more second-stage reaction wells 582.The large number of second-stage reactions available in pouch 510 allowsa robust identification strategy for organisms, illustratively bacteria,by allowing the interrogation of a number of genes for each species(illustratively housekeeping genes are used, as described below). Thegenes may be amplified in a first-stage multiplex reaction using outerfirst-stage primers targeting conserved sequences, and then subsequentlyamplified in individual second-stage reactions using inner second-stageprimers targeted more specifically to the individual species. Thisstrategy allows for the detection and identification of an unknown (andunspecified) pathogenic bacterium using a single set of assays performedin a closed system, with results available in real time. By combiningtests for a wide range of bacteria into a single assay, the problems aclinician faces when having to order individual assays for eachbacterial species can be reduced or avoided. This strategy may alsoreduce or eliminate the need for sequencing of the PCR amplicon fororganism identification.

One illustrative application of this method is the identification ofbacteria infecting infants in the first 90 days of life. Pediatricguidelines recommend aggressive evaluation of infants ≤28 days old forserious bacterial infection, including cultures for bacterial pathogensfrom blood, urine and, cerebral spinal fluid. While awaiting results ofthese cultures, which can take from 24-48 hours, infants are oftenhospitalized and placed on broad-spectrum antibiotics.

When an organism is identified, antibiotic susceptibility testing isoften required, and can be complex, particularly for organisms showingresistance to certain classes of antibiotics. For example, forGram-negative organisms showing resistance to third-generationcephalosporins, guidelines have been developed for detection of theextended-spectrum β-lactamase (ESBL) phenotype (18). However, from alaboratory standpoint, ESBL testing is labor-intensive (19), and from aclinical standpoint, such testing can be misleading, as sometimessusceptible isolates in vitro are resistant under treatment conditions(18-20). Delays in testing or misreporting of susceptibility data oftenresults in prolonged use of broad-spectrum agents or the risk ofinadequate therapy.

PCR-based detection of antibiotic resistance is becoming more common. Inparticular, detection of methicillin-resistance in S. aureus byidentification of the mecA gene has become widely accepted and is commonin clinical settings. However, despite the identification of thesegenetic determinants of antibiotic resistance, molecular testing forother resistance genes has not been widely used. This likely is due tothe complexity of the genetics. For example, over 200 ESBL genotypeshave been characterized, reflecting multiple mutations in seven or moreplasmid-encoded genes. Diagnostic strategies such as those illustratedhere, capable of high-order multiplex analysis and the flexibility tochange as the resistance landscape evolves, will aid in the developmentof rapid testing for these resistance determinants.

Currently, molecular testing is not routinely available for any of thebacterial pathogens common in young infants. Even limiting the list ofbacteria tested to that of those most likely found in these infants,this list would include 8-10 organisms, and if molecular testing wereavailable, traditional PCR methods would require a minimum of 8-10separate tests. Alternately, using currently described broad-range PCRstrategies, a sequencing step, taking almost as long as culture, wouldbe necessary for identification. A strategy such as that presented inthis Example could allow for interrogation of blood or CSF or othersterile body fluid for multiple pathogens in a single assay with resultspotentially available at the point-of-care. By way of example, 27pathogens (4 genes*27 pathogens=108 wells+12 control wells=120 totalsecond-stage reaction wells 582 in the high-density array) may be testedin a single pouch 510.

It is known that different groups of patients have different likelihoodsof infection with particular organisms based on their age, presentinglocation (for example, inside or outside the hospital), and underlyingimmunity. By designing broad-range outer primer sets for conserved genesfor first-stage PCR, the present strategy allows for the modification ofsecond-stage inner primer sets to target different populations ofpatients. In the example described above with respect to infants, innersecond-stage PCR targets illustratively would include (but not belimited to) Neisseria meningitidis, Haemophilus influenzae, Escherichiacoli, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcusagalactiae, Staphylococcus aureus and Listeria monocytogenes. It isunderstood that this list is exemplary only and that the list ofsecond-stage targets may be adjusted due to a wide variety of factors.Other populations that could be targeted include: patients in theintensive care unit, (ICU panel illustratively including Pseudomonasaeruginosa, Enterobacter cloacae, Klebsiella oxytoca and Serratiamarscescens), and patients with malignancy and immunity suppressed bychemotherapy (illustratively febrile neutropenic panel, includingopportunistic pathogens such as Staphylococcus epidermidis, Pseudomonasaeruginosa, and Viridans group Streptococci). Illustratively, in eachcase the optimized first-stage outer primer set may remain unchanged,but the panel of second-stage inner primers would be modified for thetarget population.

This strategy could also be used for identification of organisms fromculture, particularly in cases where a patient is not responding toantibiotics, and rapid organism identification might direct antibiotictherapy more appropriately, or in situations, such as possible infectionwith Neisseria meningitidis (meningococcemia) where rapid speciesconfirmation is crucial to the initiation of prophylaxis for contacts.

In addition, it is possible that this strategy may allow theidentification of novel strains of bacteria causing disease byidentifying bacteria based on a Cp and/or post-second-stageamplification melting curve “fingerprint.” Due to the broad-range designof first-stage outer primers, which are likely to amplify targets fromeven unknown bacteria, along with expected cross-reactivity of innersecond-stage primers with non-target organisms, a novel bacterium mayhave a “fingerprint” not previously encountered. It is also possiblethat different strains of the same species of bacteria might generateunique “fingerprints” (either by Cp pattern or post-second-stageamplification melting curves) and thus this technique may allowepidemiologic tracing of bacteria and outbreaks of infection. Currently,multi locus sequence typing (MLST) can be used for this purpose, butthis technique takes a significant period of time to perform. Thepresent strategy would allow for rapid identification of an outbreak,illustratively by coding instrument 800 to detect when unique Cppatterns are seen at a high frequency over a short period of time.

Theoretical Support for the Strategy:

With the advent of genomic sequencing, systematic bacteriology hasworked to come up with a definition of “species” based on genomicsequencing (Gupta. Micro and Mol Biol Rev. 1998; 62: 1435, Gupta et al.Theoretical Pop Biol. 2002; 61: 423). The “Report of the Ad HocCommittee for the Re-Evaluation of the Species Definition inBacteriology” (Stackebrandt et al. Int J Systemic and Evol Microbial.2002; 52: 1043) recommends evaluation of “protein-coding genes understabilizing selection” (housekeeping genes) for their use inphylogenetic studies to differentiate bacterial species from oneanother. The present strategy uses similar genes applied to theidentification of known and well-described pathogenic species,illustratively with results in real-time. The use of the particulargenes in the present example is further supported by other studies inwhich the utility of a range of housekeeping genes in identifying andclassifying species has been compared to previously accepted methodssuch as DNA:DNA reassociation, absolute nucleotide identity and 16S rRNAgene sequencing (Stackebrandt et al. Int J Systemic and Evol Microbiol.2002; 52: 1043, Konstantinos et al. Appl Environ Micro. 2006; 72: 7286(multiple genes), Mollet et al. Mol Micro. 1997; 26: 1005 (rpoB), Kwoket al Int J Systemic and Evol Microbiol. 1999; 49: 1181 (hsp60)).Although the present strategy bases gene selection on these types ofstudies, it is understood that other genes may be used as well.

Primer Design:

The illustrative strategy employs broad-range outer first-stage PCRprimers targeting multiple conserved protein-encoding genes andconserved non-protein-encoding sequences such as ribosomal genes.Identification of specific bacterial species is done in individualsecond-stage wells 582 of a 120 well high-density array, using specificinner second-stage PCR primers for pathogens of interest. Theillustrative broad-range outer first-stage primer sets amplify conservedgenes in phylogenetically distant bacterial pathogens. It is anticipatedthat 4-7 genes may need to be interrogated to generate a robust andspecific method for bacterial identification; it is possible that,despite directing the assays at conserved genes, a particular strain orisolate may undergo an unanticipated nucleotide sequence change in theregion of one assay, which could be rescued by a positive assay foranother gene. Illustrative initial gene targets include rpoB (RNApolymerase beta subunit), gyrB (DNA gyrase subunit B), ftsH (conservedmembrane protease), ompA (outer membrane protein A), secY (conservedmembrane translocon), recA (DNA recombination protein), groEL (hsp60heat shock protein) and the 16S rRNA gene. Primer design and initialtesting is presently underway for rpoB and gyrB.

Due to the fact that the target species come from distant arms of thebacterial phylogenetic tree, the ability to perform simple nucleotidealignment to interrogate for conserved regions is unlikely. By aligningthe genes based on their amino acid sequences, it is possible toidentify protein domains conserved across species, and it is to thenucleotide sequences of these domains that the outer first-stage primersof the nested assay are designed. By using degenerate primers, it hasbeen found that rpoB and gyrB require only 2 outer first-stage primersets (one for Gram-positive and one for Gram-negative organisms; 2-4primers per set, although it is understood that other numbers of primersets or primers per set could be used, as desired for the particularapplication) per gene to amplify all target organisms tested (14 speciesto date). It is possible that other gene targets will require a largernumber of outer first-stage primers. By limiting the number offirst-stage primer sets in the outer reaction, the multiplex of thefirst-stage PCR amplification is simplified, thus increasing the chancesof a successful assay. Further, the outer multiplex, which is often moresensitive to changes in primer number and sequence, can remainundisturbed if the panel changes, and does not need to be re-validatedfor all pathogens if one or more pathogen is added or removed. While itis desired to have a single set of first-stage primers and only changethe second-stage primers for specific panels, it is also possible thatsome panels may require unique first-stage primer sets. In the presentexample, degeneracies in the illustrative outer first-stage primer setsfor rpoB and gyrB range from 6 to 32 fold.

Illustratively, desired locations for outer first-stage primers arelocations in which two conserved regions, separated by no more thanabout 500 nucleotide base pairs, bracket a variable region with enoughsequence variability to design inner primers specific to particularbacterial species. Such locations were identified in both rpoB and gyrB,and multiple inner primer sets have been designed to the variableregions, each, for the most part, targeting a specific bacterialspecies. Where possible, the 3′-end of the primer has been designed tolie on a sequence that encodes a “signature” amino acid or acids whichis unique to the species being targeted, but different from otherbacteria. This diminishes or prevents second-stage amplification unlessthat species is present. This was not possible in all cases, and somecross-amplification is seen. Illustrative primers developed to date forrpoB and gyrB are shown in Tables 1-3.

When bacterial species are genetically too similar to be amplifiedexclusively by a unique set of primers, a “fingerprinting” techniquebased on the amplification characteristics of a set of primers can beused for species identification. An illustrative example is “enteric”organisms (e.g. E. coli, K. pneumoniae, K. oxytoca and E. cloacae) inthe rpoB gene. These enteric organisms have too much sequence similaritywithin rpoB, and designing specific inner second-stage primers hasproven difficult. Therefore, a “pan-enteric” inner second-stage primerwas designed, as well as “preferential” primers, as shown in Table 3. Anexample of how these primers would be used to generate a fingerprint isshown in FIGS. 19-20. When the preferential inner primers are used, eachenteric bacterial template shows a different pattern of crossing points,and each organism amplifies best with its own preferential inner primerset, FIG. 19A shows second-stage amplification with the pan-entericprimers. All three organisms amplified with essentially the same Cp.FIGS. 19A-19D show amplification using the preferential primers for E.coli, K. pneumoniae, and K. oxytoca and E. cloacae, respectively. Inthis illustrative example, the inner primers for K. pneumoniae have themost sequence difference, and, thus, are the most selective, with K.pneumoniae having a much earlier Cp (FIG. 19C). FIGS. 20A and 20B show achart (FIG. 20A) and decision tree (FIG. 20B) that may be used foridentification using the Cp fingerprint. As illustrated in Table 3, thebases in bold provide primer specificity. In this illustrative example,the specificity is provided by the forward primers; the reverse primerfor the E. coli, K. pneumoniae and E. cloacae assays is the same.

Enteric organisms can be differentiated from each other in the gyrBassay, as well as potentially by the melting temperature of the rpoBamplicon, depending on sequence variation. An additional strategy fordistinguishing closely-related species, for example the entericorganisms already discussed, would be to target a gene which is presentwithin that group, but less-conserved than other housekeeping genes, andnot present in other groups of bacteria. Illustratively, an outermembrane protein gene such as ompA (outer membrane protein A), presentin the outer membrane of Gram-negative bacteria, but not present inGram-positive bacteria, could be used. Outer primers would targetregions conserved within the closely-related group of organisms, andinner primers would be used to distinguish them. An illustrative set ofprimers for ompA is shown in Table 4. Thus, it is understood thatvarious combinations of first- and second-stage reactions can be used,including those identifying a particular group of organisms and othersspecific for a particular target species, for example specific bacterialspecies.

While the rpoB (RNA polymerase beta subunit), gyrB (DNA gyrase subunitB), and ompA (outer membrane protein A) genes are used in this example,it is understood that other genes may be used as well, including ftsH(conserved membrane protease), secY (conserved membrane proteintranslocon), recA (DNA recombination protein), groEL (hsp60 heat shockprotein) and the 16S rRNA gene. It is understood that these genes areillustrative only, and any combination of these and/or other genes iswithin the scope of this invention. While variability in the targetsequence at the 3′-end may be used to limit cross-amplification, it isanticipated that cross-amplification by the inner primers will occur,possibly in several genes even for the same species. This may lead to a“positive call” for more than one organism-specific assay.Identification of each organism may be made by its “fingerprint” orpattern of amplification (whether amplification occurs and Cp) over allinner primer sets.

TABLE 1  Outer First-Stage Primers: rpoB and gyrB Gene Primer SequencerpoB forward GGCTTYGARGTDCGDGACG Gram-negatives (SEQ ID NO. 1) rpoBreverse RCCCATBARTGCRCGGTT Gram-negatives (SEQ ID NO. 2) rpoB forwardBCACTAYGGBCGYATGTGTCC Gram-positives (SEQ ID NO. 3) rpoB reverseAAHGGAATACATGCYGTYGC Gram-positives (SEQ ID NO. 4) gyrB forwardTCCGGYGGYYTRCAYGG Gram-negatives-1 (SEQ ID NO. 5) gyrB forwardTCBGTNGTWAACGCCCTGTC Gram-negatives-2 (SEQ ID NO. 6) gyrB reverseRCGYTGHGGRATGTTRTTGGT Gram-negatives (SEQ ID NO. 7) gyrB forwardGGYGGWGGYGGMTAYAAGGTTTC Gram-positives-1 (SEQ ID NO. 8) gyrB forwardGGMGGYGGCGGMTAYAAAGTATC Gram-positives-2 (SEQ ID NO. 9) gyrB reverseTTCRTGMGTHCCDCCTTC Gram-positives-1 (SEQ ID NO. 82) gyrB reverseTTCATGCGTACCACCCTC Gram-positives-2 (SEQ ID NO. 10)

TABLE 2  Inner Second-Stage Primers: rpoB and gyrB Gene Primer NameSequence Target rpoB rpoB1Ents.iF04 CCGTAGYAAAGGCGAATCC Enteric(SEQ ID NO. 11) organisms rpoB rpoB1Ents.iR03 GGTWACGTCCATGTAGTCAACEnteric (SEQ ID NO. 12) organisms rpoB rpoB1Hinf.iF01ACTTTCTGCTTTTGCACGT H. influenzae (SEQ ID NO. 13) rpoB rpoB1Hinf.iR01CTTCAGTAACTTGACCATCAAC H. influenzae (SEQ ID NO. 14) rpoB rpoB1Nmen.iF01CTCATTGTCCGTTTATGCG N. meningitidis (SEQ ID NO. 15) rpoB rpoB1Nmen.iR02 TCGATTTCCTCGGTTACTTTG N. meningitidis (SEQ ID NO. 16) rpoBrpoB1Paer.iF01 TCAACTCCCTGGCGACC P. aeruginosa (SEQ ID NO. 17) rPoBrpoB1Paer.iR01 CYACGCGGTACGGGC P. aeruginosa (SEQ ID NO. 18) rpoBrpoB1Spne.iF03 AGGTTGACCGTGAAACAGG S. pneumoniae (SEQ ID NO. 19) rpoBrpoB1Spne.iF04  AGGTTGACCGTGCAACTGG S. pneumoniae (SEQ ID NO. 20) rpoBrpoB1Spne.iR03 GCTGGATACTCTTGGTTGACC S. pneumoniae (SEQ ID NO. 21) rpoBrpoB1Spne.iR04 GCTGGATATTCTTGGTTAACC S. pneumoniae (SEQ ID NO. 22) rpoBrpoB1Spyo.iF01 YGAAGAAGACGARTACACAGTT S. pyogenes (SEQ ID NO. 23) rpoBrpoB1Spyo.iR01 CGTCAACGAAATCAACAACAC S. pyogenes (SEQ ID NO. 24) rpoBrpoB1Saga.iF01 TGAAGAAGATGAATTTACAGTT S. agalactiae (SEQ ID NQ. 25) rpoBrpoB1Saga.iR01  CGTCAACAAAGTCAACAATGC S. agalactiae (SEQ ID NO. 26) rpoBrpoB1Saur.iF02  GTAAAGTTGATTTAGATACACATGC S. aureus (SEQ ID NO. 27) rpoBrpoB1Saur.iR01 CGACATACAACTTCATCATCCAT S. aureus (SEQ ID NO. 28) rpoBrpoB1Sepi.iF01  YTWGATGAAAATGGTCGTTTCYTAG S. epidermidis (SEQ ID NO. 29)rpoB rpoB1Sepi.iR01 GTTTWGGWGATACRTCCATGT S. epidermidis (SEQ ID NO. 30)rpoB rpoB1Lmon.iF01 CKAACTCGAAATTAGACGAACAA L. monocytogenes(SEQ ID NO. 31) rpoB rpoB1Lmon.iR01  TTTTCTACCGCTAAGTTTTCTGAL. monocytogenes (SEQ ID NO. 32) rpoB rpoB1Efas.iF01ACAGCYGAYATCGAAGACCA E. faecalis (SEQ ID NO. 33) rpoB rpoB1Efas.iR01ACTTCTAAGTTTTCACTTTGHGC E. faecalis (SEQ ID NO. 34) rpoB rpoB1Efas.i102ACTTCTAAGTTTTCACTTTGTAG E. faecalis (SEQ ID NO. 35) gyrB gyrB1Hinf.iF01ATTCGTCGTCAAGGTCAC H. influenzae (SEQ ID NO. 36) gyrB gyrB1Hinf.iR01CGATTGAGGCTCCCCTAAA H. influenzae (SEQ ID NQ. 37) gyrB gyrB1Ecol.iF01GTTATCCAGCGCGAGGGTA E. coli (SEQ ID NO. 38) gyrB gyrB1Ecol.iR01 GTGCCGGTTTTTTCAGTCT E. coli (SEQ ID NO. 39) gyrB gyrB1Kpne.iF01GATAACAAAGTTCACAAGCAGATG K. pneumoniae (SEQ ID NO. 40) gyrBgyrB1Kpne.iR01 GTCGGTTTCGCCAGTCA K. pneumoniae (SEQ ID NO. 41) gyrBgyrB1Koxy.iF02 GTTTCTGGCCGAGCTAYGA K. oxytoca (SEQ ID NO. 42) gyrBgyrB1Koxy.iR02 CGTTTTGCCAGRATYTCGTAT K. oxytoca (SEQ ID NQ. 43) gyrBgyrB1Eclo.iF01 TATCCAGCGCGAAGGCA E. cloacae (SEQ ID NO. 44) gyrBgyrB1Eclo.iR01 GGCCAGAAACGCACCAT E. cloacae (SEQ ID NO. 45) gyrBgyrB1Nmen.iF01 AACACTTCGTCCGCTTCGT N. meningitidis (SEQ ID NO. 46) gyrBgyrB1Nmen.iR01 GCGAGGAAGCGCACGGT N. meningitidis (SEQ ID NO. 47) gyrBgyrB1Paer.iF01 TCGCCACAACAAGGTCTG P. aeruginosa (SEQ ID NO. 48) gyrBgyrB1Paer.iR01 GGCCAGGATGTCCCARCT P. aeruginosa (SEQ ID NO. 49) gyrBgyrB1Saur.iF01  GTCATTCGTTTTAAAGCAGATGGA S. aureus (SEQ ID NO. 50) gyrBgyrB1Saur.iR01 GTGATAGGAGTCTTCTCTAACGT S. aureus (SEQ ID NO. 51) gyrBgyrB1Spne.iF01 AGAATACCGTCGTGGTCAT S. pneumoniae (SEQ ID NO. 52) gyrBgyrB1Spne.iR01 ACCTTGGCGCTTATCTGT S. pneumoniae (SEQ ID NO. 53) gyrBgyrB1Spyo.iF01 AACGACTCAGTTTGATTACAGTG S. pyogenes (SEQ ID NO. 54) gyrBgyrB1Spyo.iR01 AAATGTTCTTCTTGTTCCATACCT S. pyogenes (SEQ ID NO. 55) gyrBgyrB1Saga.iF01  GYAAGGTTCATTATCAAGAATAYCAA S. agalactiae (SEQ ID NO. 56)gyrB gyrB1Saga.iR01 AAGTGAACTGTTGTTCCTGATAAG S. agalactiae(SEQ ID NO. 57) gyrB gyrB1Sepi.iF01 GCTATTCGATTCAAAGCCGATAAS. epidermidis (SEQ ID NO. 58) gyrB gyrB1Sepi.iR01 TCTAACTTCCTCTTCTCTTTCATCTTT S. epidermidis (SEQ ID NO. 59) gyrBgyrB1Efas.iF01 CGTCGTGAAGGACAAGAAGATAAA E .faecalis (SEQ ID NO. 60)gyrB  gyrB1Efas.iR01 CTTGTTGCTCTCCTTCAATG E. faecalis (SEQ ID NQ. 61)gyrB gyrB1Lmon.iF03 CCAATTTATTTGGAAGGTGAACG L. monocytogenes(SEQ ID NO. 62) gyrB gyrB1Lmon.iR05 GCGAATGAAATGATRTTGCTTGAGAL. monocytogenes (SEQ ID NQ. 63)

TABLE 3  Inner Primers for Enteric Organisms in rpoB Preferential TargetPrimer Sequence “pan-enteric” rpoB1Ents.iF04 CCGTAGYAAAGGCGAATCC(SEQ ID NO. 11) rpoB1Ents.iR03 GGTWACGTCCATGTAGTCAAC (SEQ ID NO. 12)E. coli rpoB1Ecol.iF01 ACTCCAACCTGGATGA A GA (SEQ ID NO. 64)rpoB1Ents.iR02 ACGTCCATGTAGTCAACC (SEQ ID NO. 65) K. pneumoniaerpoB1Kpne.iF01 GAACTCCAACCTGGATGAAA A C  (SEQ ID NO. 66) rpoB1Ents.iR02ACGTCCATGTAGTCAACC (SEQ ID NO. 65) E. cloacae/ rpoB1Eclo.iF01ACTC CAACCTGGATGA C GA K. oxytoca (SEQ ID NO. 67) rpoB1Ents.iR02ACGTCCATGTAGTCAACC (SEQ ID NO. 65)

TABLE 4  Outer and Inner Primers for Enteric Organisms in ompA TargetPrimer Sequence Outer primers Enterics ompA1Ents.oF_aTCGCYACCCGTCTGGAATA (SEQ ID NO. 68) ompA1Ents.oR_a CGTCTTTMGGRTCCAKGTTG(SEQ ID NO. 69) Inner primers E. coli ompA1Ecol.iF_aGTCTGGAATACCAGTGGACC (SEQ ID NO. 70) ompA1Ecol.iR a CTGATCCAGAGCAGCCT(SEQ ID NO. 71) K. pneumoniae  ompA1Kpne.iF_a CGGTCAGQAAQATGCTGC(SEQ ID NO. 72) ompA1Kpne.iR_a GGTGAAGTGCTTGGTAGCC (SEQ ID NO. 73)K. oxytoca ompA1Koxy.iF_a CGGTCAGGAAGATGTTGCT (SEQ ID NO. 74)ompA1Koxy.iR_a TGACCTTCTGGTTTCAGAGT AGAT (SEQ ID NO. 75) E. cloacaeompA1Eclo.iF_a AACATCGGCGACGGCAA (SEQ ID NO. 76) ompA1Eclo.iR_aGCTGGAGCAACGATTGGC (SEQ ID NQ. 77)

As discussed above, while ideally each organism would be amplifiedexclusively by its appropriate inner second-stage primer set, crossamplification can occur. Due to expectations that organisms will showmultiple, but predictable, amplification curves in a single conservedgene (see the example with K. oxytoca and H. influenzae rpoB primers inFIGS. 15A-15C described below), use of supervised learning isanticipated for the instrument 800 software, based on the pattern ofamplification curves generated by the panel of conserved gene assays.For each bacterial species, clinical isolates whose identity has beenconfirmed by biochemical identification and, where appropriate, genespecific DNA sequencing, will form the basis for training data set forthe predication software. Depending on the characteristics of the data,it is anticipated that either a nearest-neighbor algorithm or aPrinciple Component Analysis based ranking system will be used togenerate an identity “fingerprint” for each target bacteria.

Given the complexity of a nested multiplex amplification reaction,considerable optimization may be required. In one illustrative method,outer and inner primer sets are initially tested separately in simplePCR reactions (one primer set, one template) to demonstrate whether eachprimer set will generate a template-dependent real-time amplificationcurve, with a resultant PCR product of the expected size.Illustratively, size may be demonstrated by agarose gel electrophoresis,although other methods are known in the art and are within the scope ofthis disclosure. Further assay optimization illustratively takes placeusing nested PCR and real-time detection (for example, amplifiedproducts may be detected using a fluorescent double-stranded DNA bindingdye).

The fluorescence crossing point (Cp), of the nested reaction providesrelative quantitation of the initial target, and can also be used as ameasure of the efficiency of the PCR reactions. Comparison of Cps allowsevaluation of assays run under different conditions. Both inner andouter primer sets may be optimized to generate early nested Cps,representing efficient PCR. Other parameters examined in optimizationmay be the Cp of the reaction when no template is added and the Cp ofthe reaction when human genomic DNA is used as a template. Productsformed in the no-template reaction are non-specific and likely representprimer dimers. Products in the human genomic reaction that are notpresent in the no-template reaction represent cross-reaction with humansequences, and may interfere with specificity when the assay isperformed directly from clinical specimens. In both cases, theamplification curves are false-positives, and primer sets with Cps of,illustratively, less than 32 cycles in these reactions are redesigned.While 32 cycles is chosen as the Cp cut-off point, it is understood thatother crossing points could be chosen, depending on the desiredspecificity and sensitivity.

In the illustrative example, once assays have been optimized insingleplex format, assays may then be multiplexed and tested for limitof detection and specificity (amplification of only the intendedtarget), with comparison to the singleplex assays. Assays may beredesigned as necessary. During multiplex optimization, assays may bemoved to instrument 800, so that optimization may occur under theconditions of the final assay.

Two sets of experiments demonstrating the illustrative bacterialidentification strategy are shown. FIGS. 15A-15C show an experiment inwhich both rpoB and gyrB are used for identification and differentiationof Gram-negative organisms. Bacteria from different phyla have beenincluded to help display the broad range of the outer first-stageprimers, and because they are important antibiotic-resistantGram-negative pathogens that could be targeted in an illustrative assayaccording to the methods discussed herein. In the experiment shown inFIGS. 15A-15C, DNA was extracted from clinical isolates of the selectedorganisms. Total bacterial nucleic acid was amplified in a multiplexouter first-stage PCR reaction containing primer sets targeting both therpoB and gyrB genes of Gram-negative organisms (one degenerate primerpair per gene). All first-stage amplicons were then nested into allinner second-stage PCR reactions each containing a primer set specificfor a particular organism and amplified in the presence of LC Green®Plus, although other detection means may be used, as described above. Asshown in FIGS. 15A-15C, although first-stage amplicons for eachbacterium are tested with all inner second-stage primer sets, the onlyinner primer sets showing significant amplification are those directedat the target organism. This demonstrates the specificity of the innersecond-stage assay. FIGS. 16A-16D show an experiment looking at the rpoBgene, in this case with Gram-positive targets. As was seen in FIGS.15A-C, the only inner second-stage primer set showing significantamplification is that directed at the target organism. FIGS. 17A and 17Bshow amplicon melting profiles for the second-stage amplicons generatedin the experiment shown in FIGS. 15A-15C. As can be seen in FIGS. 17Aand 17B, the amplicons melt with characteristic profiles, which could beused for further specificity in organism identification. Subsequently,second stage primers have been developed for S. epidermidis, K. oxytoca,and E. faecalis for use with the same first-stage primers.

The above examples were done as individual reactions, for the purposesof optimizing. FIGS. 18a-b show amplification with a first-stageamplification and second-stage amplification done in an instrument 800.Inner second-stage primers were spotted in triplicate in second-stage581. The replicates were spotted in locations remote from each other, tominimize positional effects. The results shown in FIG. 18A are for theN. meningitidis gyrB assay. The three amplification curves with thelowest Cp (about 18-20) are the neisseria amplification curves, whilethose coming up later (Cp>25) show primer-dimers or other non-specificamplification. FIG. 18B shows the results for the N. meningitidis rpoBassay, confirming the results from the gyrB gene, with even greaterseparation between the target amplification and the non-specificamplification. In addition to Cp, the results may be confirmed usingmelting curve analysis. Similar results have been obtained with othertargets. Clinical samples have been run on this panel, resulting in 0-4positive targets per sample.

It is understood that while the above examples are used for identifyingbacterial species, the method could be applicable to identification ofunknown bacteria. Illustratively, second stage inner primers could bedesigned more generally, and spread throughout the “sequence space” ofthe variable inner region. Bacterial identification would then be basedon the Cp “fingerprint” across the second stage PCR array. The bacterialtraining set described above would be used to determine fingerprints forknown bacterial species, and additional species/novel fingerprintsencountered over time could be added to the data.

Additionally, while the above examples focus on identifying bacterialspecies, a similar method could be used to identify antibioticresistance, with or without species identification. Many antibioticresistance genes are found on plasmids, and for treatment purposes, thepresence or absence of the gene or gene mutation may be more importantthan identification of the pathogen. As an illustrative example, onecould test for one or more of the TEM, SHV, and CTX-M β-lactamase genesfound on plasmids in Gram-negative enteric pathogens. In this example,outer primers would target conserved portions of the plasmid; innerprimers could also target conserved regions when the presence or absenceof the plasmid confers resistance (e.g. ampicillin resistance), oralternatively could target regions in which mutations conferringdifferent resistance properties (e.g. the extended-spectrum β-lactamasephenotype) are found. If point mutations lead to a change in theresistance pattern, probes can be included in the second stage reactionto identify the mutation, illustratively by probe melting. The probesmay be labeled, or the second-stage reaction may include a dsDNA bindingdye, illustratively a saturation dye (see, e.g., U.S. patent applicationSer. No. 11/485,851, herein incorporated by reference). Table 5 showsillustrative probes targeting several mutations in the TEM β-lactamasegene.

TABLE 5 WT Mutant Probe codon codon TEM  ACTTGGTTgAGTACTCACCAGTC GAG(104) wt ACAGAA (SEQ ID NO. 78 TEM  ACTTGGTTaAGTACTCACCAGTC GAG aAG(104) EK ACAGAA (SEQ ID NO. 79) TEM  CCTTGATcGTTGGGAACCGGAGC CGT(164) wt TGAAT (SEQ ID NO. 80) TEM  CCTTGATaGTTGGGAACCGGAGC CGT aGT(164) RS TGAAT (SEQ ID NO. 81)

Further, it is understood that the methods described herein could beused for the identification of other, non-bacterial species. Asdescribed above, the outer first-stage primers may be targeted togenerally conserved regions of target genes, while the innersection-stage primers would be targeted to sequences specific for thespecies or genus. In one example, the methods described herein could beapplied to fungal identification. Such an application of the presentinvention could significantly reduce the time to diagnosis, assporulation, which can take several days to several weeks, is currentlyused to identify a fungal species. In some cases, a fungus may notsporulate at all in the clinical laboratory, and would only beidentifiable by molecular methods. If four conserved genes are requiredfor the outer first-stage amplification, twenty-five or more innersecond-stage targets may be tested per gene, assuming approximately 100second-stage wells 582. Other non-limiting potential combinations are asfollows:

8 genes×12 inner primers=96 reactions

7 genes×14 inner primers=98 reactions

6 genes×16 inner primers=96 reactions

5 genes×20 inner primers=100 reactions

It is understood that these combinations are illustrative only, and thatother combinations are possible. For example, if there are more secondstage wells 582 in high density array 581, then additional genes and/ormore inner primer sets may be used. Alternatively, if the first-stageamplicon is large, it may be desirable to amplify several regions of thefirst-stage amplicon, illustratively for better identification ofstrains of the same organism. If desired, amplicon melting temperaturesand/or probes may be used for further identification or for confirmationof results. Illustratively, the second-stage amplicons includesufficient information for identifying at least 50% of the species orstrains, and more illustratively 90% of the species or strains.

EXAMPLE 5: RAPID EPIDEMIOLOGIC TYPING OF BACTERIA

In this example, high-order multiplex PCR testing for identification andcharacterization of bacteria, illustratively MRSA is described. Whilereference is made to bacteria generally and MRSA specifically, it isunderstood that these examples are illustrative only, and the methodsdescribed herein have other applications. In the course of developingthe nmPCR assays for the bacterial identification panel described above,a protocol has been established for designing the primer sets, testingthem, revising as needed and testing in singleplex and multiplexformats.

Currently, it is anticipated that 68 primers may be used for theidentification and characterization of MRSA isolates in the illustrativeexample (Table 6).

TABLE 6 Proposed Targets for the MRSA panel #Outer primers ApplicationGene Full Name or function in multiplex Identification of rpoB RNApolymerase beta subunit 2 S. aureus, gyrB DNA gyrase subunit b 2Detection of coagulase- rpoB RNA polymerase beta subunit 2 negativestaph gyrB DNA gyrase subunit b 2 Methicillin resistance mecA EncodesPBP2A 2 mecA/orfX links mecA to S. aureus 2 Other drug ermA, ermB,Macrolide resistance 6 susceptibility ermC (inducible clinda resistance)msrA Macrolide resistance 2 (clinda sensitive phenotype) dfrATrimethoprim-sulfa resistance 2 tetK, tetM Tetracycline resistance 4mupR Mupirocin resistance 2 vanA, vanB, Vancomycin resistance 6 vanZ PVLlukF, lukS Panton-Valentine leukocidin 2 SCCmec typing I-J1 region 2V-ccr complex 2 III-J3 region 2 V-J1 region 2 I, II, IV, VI-J3 region 2II, IV-ccr complex 2 II-J1 region 2 III-J1 region 2 II, III-mec complex2 MLST arcC Carbamate kinase 2 aroE Shikimate dehydrogenase 2 glpFGlycerol kinase 2 gmk Guanylate kinase 2 pta Phosphateacetyl transferase2 tpi Triosephosphate isomerase 2 yqiL Acetyle coenzyme A 2acetyltransferase 2 Total Outer Primers: 68

For initial design and testing, cultured bacterial isolates of knowngenotype will be used as targets. To prepare the assay for clinical use,limit of detection studies will be performed, as well testing of sampleswith other organisms to demonstrate sufficient sensitivity andspecificity for direct-from-specimen testing. Anticipated clinicalspecimen types include sputum, a nasal swab, a nasal aspirate, atracheal aspirate, another respiratory specimen, a wound culture, and asterile site culture (illustratively blood, spinal fluid, joint fluid,pleural fluid, etc.), although other specimen types may be used.

In the illustrative S. aureus-specific testing proposed herein, outerprimers will be designed to be specific for S. aureus, decreasing thepossibility of amplifying other bacteria. In addition, assays for S.epidermidis and other coagulase-negative staphylococci (“CONS”) may beincluded, to detect these as potential contributors to mecAamplification. mecA identification by PCR is well-established (23-26),and nmPCR assays may be based on published primers or may be designedanew. Published assays target various regions of mecA, with a range ofamplicon sizes. It is understood, however, that certain assays may needto be re-designed, particularly if pairs of primers interfere duringfirst-stage PCR or are not compatible with the amplification conditions.Illustratively, assays will be chosen that best fit with theamplification conditions of instrument 800.

Assays have been published and at least one has been FDA approved forthe direct identification of MRSA from clinical specimens, using an S.aureus-specific sequence from orfX, an open reading frame of unknownfunction at the chromosomal right junction where SCCmec integrates intothe S. aureus genome (23, 24). Sequences within this gene illustrativelymay be used for the reverse PCR primer, as mecA is reliably linked tothe S. aureus-specific orfX sequence. Thus, by providing one primer inthe mecA sequence and the other primer in the orfX sequence,misclassification due to the presence of mecA-containing CONS within asample may be avoided. Since the amplicons illustratively will be S.aureus-specific, nesting the second-stage primers is optional. However,nesting may be desired, particularly when generating shortersecond-stage amplicons for the melting analysis discussed below.

It is known that S. aureus contains numerous virulence factors thatenhance its pathogenicity. Many virulence factors are present in mostisolates. However, there are some factors for which an association withpathogenicity is assumed, but not definitive (27, 28). A specificexample is the Panton-Valentine leukocidin, present in the most virulentCA-MRSA strains and thought to lead to the highly invasive nature ofthis pathogen (27, 29, 30). Routine and unbiased testing could establishthe role of PVL in clinical disease, and thus PVL testing may beincluded in the illustrative assay. The initial illustrative design willbe based on well-characterized assays targeting the lukS-PV and lukF-PVgenes (31, 32).

In addition, nmPCR assays may be included for the detection of knowngenes conferring resistance to anti-staphylococcal agents such astetracyclines, macrolides and lincosamides (see Table 6). vanA, vanB andvanZ are targets for detecting vancomycin resistance (VRSA) (33, 34). Animportant advantage of the instrument 800 is its flexibility; new testscan be added to the first-stage multiplex assay, and inner primers maybe modified as new resistance genes emerge. It is anticipated that thesusceptibility portion of the assay will evolve as new mechanisms aredetected, to include resistance determinants for VISA (vancomycinintermediate S. aureus), as well as newer agents such as linezolid anddaptomycin.

SCCmec typing is integral to MRSA clone identification (35, 36). Thereare currently 5 major SCCmec types described, as defined by mec complexclass and ccr allotype; SCCmec subtypes are defined by variations in theJ region (J1-J3) (35, 37). A number of PCR-based SCCmec typing methodshave been described, including multiplex strategies which candifferentiate the major types (38-41). A widely used multiplex is thatof Oliveira and Lancaster (40) herein incorporated by reference,recently updated to include assays for SCCmec types IV and V (42). It isexpected that a version of this assay will be adapted for instrument800. This illustrative multiplex, using 20 primers to amplify 10regions, identifies SCCmec type based on the presence of specific bandson a gel, corresponding to the presence or absence of amplificationwithin a specific well 582. The genetic organization of SCCmec andsimilarities between the 5 types may pose difficulties for the design ofnested primers. Accordingly, sequence alignments will be used todetermine appropriate unique sites either inside or outside thepublished primers. Additionally, the ccr region is not interrogated inthis illustrative multiplex. Primers for this region may need to beadded to define novel SCCmec types. Other multiplex methods have beenpublished (38-41), including a recent paper describing a multiplexSCCmec PCR, using only eight primers to amplify and distinguish the fivemajor SCCmec types (38). These assays could be substituted or added in alater embodiment.

Current culture-based bacterial detection strategies take several days,and do not provide data regarding the clonality of isolates. Identifyingclonality is an important step in outbreak identification and tracking.The ability to conduct rapid diagnostic testing—with the power toprovide simultaneous typing of bacterial clones—would significantlyimprove patient care and infection control, and improve ourunderstanding of both hospital and community risks. Common methods foroutbreak detection and clone-typing of bacterial isolates arepulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing(MLST). As currently performed, however, both PFGE and MLST are costly,time-consuming, and available primarily for research purposes. Bothmethods are primarily performed when an outbreak is already suspected,and essentially provide confirmatory data, rather than true detection.Development of the assays described herein, which illustratively usehigh-resolution melting, along with heteroduplex analysis, would allowrapid, modest cost, real-time isolate typing that could become standardfor every bacterial isolate.

Conventional MLST requires sequencing of multiple amplicons (43). Evenwith automation, this introduces expense, delay and complexity.Second-stage amplification, in conjunction with high-resolution melting,as described in U.S. Patent Publication Nos. 2005-0233335 and2006-0019253, herein incorporated by reference, along with heteroduplexanalysis (described below) may be used for typing strains,illustratively of S. aureus isolates. A recent paper described a methodfor MLST analysis of S. aureus using a microchip, supports thefeasibility of performing such typing without sequencing (21).

Melting curve analysis may be used to characterize products that areamplified. A double-stranded DNA binding dye, added to the PCR reactionto monitor amplification, can also be used to determine thecharacteristic temperature at which 50% of the DNA product denatures(the amplicon “Tm”). High-resolution melting, as described in U.S.Patent Publication Nos. 2005-0233335 and 2006-0019253, is extremelysensitive to changes in nucleotide sequence, and allows mutationscanning and genotyping (44, 45). Properties of the specialized dye usedin high resolution melting, such as LC Green® Plus (Idaho Technology),instrumentation, and software allow even single base-pair changes in PCRproducts to be detected by changes in the shape of the melting curve(46). In a diploid genome, homozygous changes in nucleotide compositioncan be difficult to detect in a PCR amplicon, even using high-resolutionmelting. Simpler to identify are heterozygous changes, in which DNA“heteroduplexes” are formed during melting (47). In such analysis,amplicons from the two different alleles are allowed to pair, withmismatches at the heterogeneous sites. This leads to destabilization ofthe duplex, and an early melt transition when compared to controls. Thismethod is used frequently in human genetics for detection of singlenucleotide polymorphisms (SNPs) (44, 47, 48). An example of such meltingcurve analysis is shown in FIGS. 21A and 21B.

For the detection of changes in a haploid bacterial genome, heteroduplexanalysis can also be used. Illustratively, a control or standard DNA ofknown sequence is added to the PCR reaction prior to amplification. Bothcontrol and unknown samples are amplified and analyzed by melt. Anychanges in the unknown sample relative to the control are detectable bythe formation of heteroduplexes, and the presence of early-meltingamplicons. In the illustrative embodiment, high resolution melting andheteroduplex analysis is used for typing analysis of S. aureus.Amplification of seven published MLST genes (arcc, aroe, glpf, gmk, pta,tpi, yquil) will be performed by nmPCR, illustratively with thepublished primers (49) used in the outer reaction, and two sets of innerprimers will span the amplicon so that all potential polymorphic sitesare interrogated. A schematic of the inner PCR is shown in FIG. 22.Standards, consisting of known sequenced alleles for each gene (up to 33alleles/per gene have been described to date), will be added to singlewells 582 with appropriate primers. “Sample-only” wells (no standard)will be present to characterize the unknown sample. During PCR,sample-only wells will amplify only the DNA of the unknown, while thewells with standards will amplify both. Where the unknown differs fromthe standard allele, heteroduplex DNA will be formed, and meltdifferences will be detectable when compared to the sample-only well.Where the unknown and the standard allele are the same the melts will beidentical, thus identifying the allele. It is understood that strainshaving differences from all alleles represented in the array will beidentified only as “different” and may require sequencing to define.

The feasibility of this approach was tested using clinical isolates withdata shown in FIGS. 23A and 23B. FIG. 22 shows a simplified schematic ofhow the second-stage amplification could be used for this typinganalysis. Only six wells 582 are shown. The first panel shows a singleMLST gene (“Gene 1”) that has 5 “standard” alleles (A-E) represented.The top well does not have a standard so that the unknown will beamplified and melted alone. In the second panel, the outer amplicon fromunknown “X” enters the array and is distributed to all wells. In theuppermost well, X alone is amplified; in each of the lower wells, X andone of the standard alleles are amplified together. After PCR, theamplicons undergo high-resolution melt analysis, illustratively withcurves normalized to the melting curve of X. Where X and the standardallele are different, heteroduplexes form, and the melt does not matchthat of X. Where X and the standard are the same, the melting curvesmatch, and the allele is identified. In the example shown here, thecurves for X and for C have been found to be the same, thus X=C.Preliminary data supporting this approach is shown in FIGS. 23A and 23B.It is expected that to test every target allele for S. aureus in thismanner, 162 or more wells 582 will be needed. It is understood that thismethod is particularly well suited for targets that are haploid, asidentity is determined by matching melting curves. While this method canbe applied to diploid or polyploid targets, the analysis would be morecomplex.

The above example for S. aureus strain typing primarily uses genomictarget sequences. It is understood that plasmid targets may also beimportant. For example, resistance to various antibiotics can beconferred by genes that are carried on plasmids. It is known that smalldifferences in these sequences can affect the resistance conferred. Insuch an example, the exact species may be less important thanidentifying the plasmid-based gene, in which case the second-stageamplification and melting may focus primarily or exclusively on genescarried by plasmids. Accordingly, plasmid target sequences may be testedin a similar manner, with or without identifying the species or thestrain.

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While references are made herein to PCR, it is understood that thedevices and methods disclosed herein may be suitable for use with othernucleic acid amplification or other biological processing methods, asare known in the art, particularly methods that benefit from afirst-stage multiplex reaction and a second-stage individual reaction.Illustrative non-limiting second-stage reactions include primerextension, including allele-specific primer extension; extensionterminations, including termination by incorporation of one or moredideoxy nucleotides; incorporation of fluorescent or non-fluorescentlabels; and other enzymatic reactions requiring a change in reactionmixture components or component ratios, such as asymmetric PCR,allele-specific PCR, invader assays, and other isothermal amplificationor detection chemistries.

Although the invention has been described in detail with reference topreferred embodiments, variations and modifications exist within thescope and spirit of the invention as described and defined in thefollowing claims.

What is claimed is:
 1. A method for determining a crossing point (Cp)for each of second-stage amplicons in each of second-stage reactionwells from each of amplification curves, comprising the steps of:obtaining a sample comprising a target nucleic acid of a specificspecies or strain of a bacterium, amplifying the target nucleic acidthat is present in the sample in a first amplifying step, wherein saidfirst amplifying step is performed in a reaction mixture comprising thetarget nucleic acid and a pair of first-stage primers, wherein eachprimer of the pair of first-stage primers hybridizes to a conservedregion of a bacterial gene shared by various different bacterialspecies, thereby generating an amplified reaction mixture comprising afirst-stage amplicon, dividing the amplified reaction mixture into a setof second-stage reaction wells, wherein the set of second-stage reactionwells comprises a plurality of the second-stage reaction wells, whereineach of the second-stage reaction wells of the plurality of thesecond-stage reaction wells comprises a different pair of second-stageprimers configured to amplify the first-stage amplicon, and at least oneprimer of the different pair of second-stage primers in each of thesecond-stage reaction wells only specifically hybridizes to a variableregion of the bacterial gene of one specific species or strain ofbacterium of the various different bacterial species and wherein the atleast one primer of the pair of second-stage primers is a nested primerof the first-stage primers, amplifying the amplified reaction mixture ineach of the second-stage reaction wells by subjecting each of thesecond-stage reaction wells to amplification reactions in the presenceof a fluorescent dye, thereby producing a plurality of second-stageamplicons in each of the second-stage reaction wells, wherein thesecond-stage amplicons comprise the nucleic acid sequences of thesecond-stage primers and the fluorescent dye, generating a plurality ofamplification curves of the amplification reactions, wherein each of theamplification curves is generated by plotting the intensity offluorescence generating from the fluorescent dye versus numbers ofcycles of one of the amplification reactions, and determining thecrossing point (Cp) for each of the second-stage amplicons in each ofthe second-stage reaction wells from each of the amplification curves.2. The method of claim 1, wherein the bacterial gene is a housekeepinggene.
 3. The method of claim 1, wherein the reaction mixture furthercomprises an additional pair of primers, wherein each primer of theadditional pair of primers hybridizes to a different conserved region ofa different target nucleic acid from the specific species or strain ofthe bacterium that may be present in the sample and the differentconserved region is shared by various different bacterial species orstrains, thereby generating an additional first-stage amplicon in theamplified reaction mixture, and the dividing step further comprisesdividing the amplified reaction mixture into an additional set of thesecond-stage reaction wells, wherein the additional set of thesecond-stage reaction wells comprises a plurality of additional reactionwells, wherein each well of the plurality of the additional reactionwells comprises a different pair of primers configured to amplify theadditional first-stage amplicon, and at least one primer of thedifferent pair of primers in each of the additional reaction wells onlyspecifically hybridizes to a variable region of the bacterial gene ofone specific species or strain of bacterium of the various differentbacterial species or strain, wherein the different pair of primers isdifferent than the different pair of second-stage primers.
 4. The methodof claim 3, wherein the different pair of the second-stage primers ineach of the reaction wells comprises a first primer and a second primerand the first primer hybridizes to the variable region of the bacterialgene of one specific species or strain of bacterium of the variousdifferent bacterial species or strain.
 5. The method of claim 4, whereinthe first primer in a first reaction well of the plurality of the secondstage reaction wells comprises a nucleic acid sequence, wherein a 3′-endof the nucleic acid sequence only specifically hybridizes with a nucleicacid sequence of a specific species or strain of bacterium of thevarious different bacterial species or strain, wherein the nucleic acidsequence of the specific species or strain of bacterium encodes aminoacids which are unique to the specific species or strain of bacteriumsuch that the amplification reactions in the first reaction well of theplurality of the second stage reaction wells are prevented in conditionsof the amplification reactions unless the target nucleic acid of thespecific species or strain of the bacterium is present in the sample.